Alteration of immunoglobulin glycosylation correlates with inflammatory diseases and infectious diseases including parasitic infections. Immunoglobulin glycosylation patterns may be implicated in disease development and have also been proposed as diagnostic tools for several diseases. Previous studies have reported the immunoglobulin profiles in experimental animals and in patients infected with the carcinogenic human liver fluke, Opisthorchis viverrini. However, the N-glycosylation profiles of immunoglobulins and their subclass-specific glycoforms in opisthorchiasis patients have never been elucidated. Here, N-glycosylation patterns of immunoglobulins and their subclass-specific glycoforms in sera of O. viverrini-infected patients were investigated using triple quadrupole mass spectrometry coupled with multiple reaction monitoring. Peptide fragmentation was utilized to quantify the immunoglobulin glycoforms normalized to the unique peptide of each subclass. Overall, serum levels of IgG and IgA in O. viverrini patients were significantly increased compared to uninfected controls. Twenty-seven glycoforms were detected based on analysis of detached glycans in all immunoglobulin subclasses. The abundance of immunoglobulin glycopeptides in serum of opisthorchiasis patients deviated significantly from controls. Immunoglobulin glycosylation patterns were associated with both pro- and anti-inflammatory properties. In conclusion, O. viverrini infection alters the serum immunoglobulin glycosylation profile and these changes could distinguish between O. viverrini-infected individuals and healthy controls. SIGNIFICANCE: We demonstrated that both quantities and glycoforms of serum immunoglobulin subclasses were altered in Opisthorchis viverrini-infected individuals as investigated by the QqQ-MS-MRM method. Patterns of immunoglobulin with a specific glycoform might contribute to immune responses to O. viverrini infection.

Functional Proteomics aims to the identification of in vivo protein-protein interaction (PPI) in order to piece together protein complexes, and therefore, cell pathways involved in biological processes of interest. Over the years, proteomic approaches used for protein-protein interaction investigation have relied on classical biochemical protocols adapted to a global overview of protein-protein interactions, within so-called "interactomics" investigation. In particular, their coupling with advanced mass spectrometry instruments and innovative analytical methods led to make great strides in the PPIs investigation in proteomics. In this review, an overview of protein complexes purification strategies, from affinity purification approaches, including proximity-dependent labeling techniques and cross-linking strategy for the identification of transient interactions, to Blue Native Gel Electrophoresis (BN-PAGE) and Size Exclusion Chromatography (SEC) employed in the "complexome profiling", has been reported, giving a look to their developments, strengths and weakness and providing to readers several recent applications of each strategy. Moreover, a section dedicated to bioinformatic databases and platforms employed for protein networks analyses was also included.

To reveal calcium-mediated germination in soybean, a gel-free/label-free proteomics was performed in radicle of seed imbibed with CaCl2. Morphological analysis presented promoting and suppressing performance of seed growth under 5 and 50 mM CaCl2, respectively. A total of 106 and 581 proteins were identified in response to 5 and 50 mM CaCl2, respectively. Among 33 proteins, which were simultaneously affected by 5 and 50 mM CaCl2 imbibition, proteins related to protein metabolism, cell, development, and stress showed reversed abundance in response to CaCl2 on dose-dependent manner. Notably, protein abundance of late embryogenesis abundant (LEA) 4-5, LEA4, and dehydrin decreased and increased by 5 and 50 mM CaCl2, respectively, consistent with the transcript level. Moreover, inhibited biosynthesis of gibberellic acid repressed growth of 5 mM CaCl2-imbibed soybean, while inhibition of abscisic acid biosynthesis released the suppressing effects of 50 mM CaCl2. Taken together, these results suggest that decreased or increased protein abundance of LEA4-5, LEA4, and dehydrin might determine promoting or suppressing effects of low or high level of calcium on soybean through enhancing seed sensitivity to gibberellic acid or abscisic acid during radicle protrusion. SIGNIFICANCE: Calcium serves as a versatile signal in plant growth; however, calcium-mediated germination on dose-dependent manner remains elusive. In this study, dual effects of calcium on radicle protrusion in soybean were investigated using proteomic approach. Radicle growth of germinating seed was improved by 5 mM CaCl2; however, it was retarded by 50 mM CaCl2. Late embryogenesis abundant (LEA) 4-5, LEA4, and dehydrin displayed converse profiles in response to low and high concentrations of CaCl2 at both protein abundance and gene expression level. Inhibited biosynthesis of gibberellic acid (GA) significantly impeded radicle protrusion in presence of low concentration of CaCl2, while inhibiting of abscisic acid (ABA) biosynthesis released suppression induced by high concentration of CaCl2. These findings suggest that LEA proteins are associated with calcium-mediated radicle protrusion on dose-dependent manner, and seed sensitivity to GA and ABA might determine promoting and suppressing effects of calcium on radicle protrusion in soybean.

The term proteoform describes all combinations of change in a protein, as elucidated through intact mass proteomics. Paleoproteomic studies have begun using digestion-free and top-down techniques to access information from ancient and historical remains. However, to discuss protein changes that uniquely occur to archaeological and paleontological proteomes as the result of diagenesis (i.e., physical and chemical change imparted by burial), a novel term is needed that both addresses issues of combinatorics and distinguishes diagenetic-specific alteration. SIGNIFICANCE: The term diagenetiform provides the opportunity to communicate clearly the sets of diagenetic changes found on preserved proteins. The diagenetiform nomenclature will allow for top-down paleoproteomic studies to accurately describe the total changes detected on ancient proteins.

Ancient proteomics is being applied to samples dating further and further back in time, with many palaeontological specimens providing protein sequence data for phylogenetic analysis as well as protein degradation studies. However, fossils are a precious material and proteomic analysis is destructive and costly. In this paper we consider three different techniques (ATR-FTIR, MALDI-ToF MS and chiral AA analysis) to screen fossil material for potential protein preservation, aiming to maximise the proteomic information recovered and saving costly time consuming analyses which may produce low quality results. It was found that splitting factor and C/P indices from ATR-FTIR were not a reliable indicator of protein survival as they are confounded by secondary mineralisation of the fossil material. Both MALDI-ToF MS and chiral AA analysis results were able to successfully identify samples with surviving proteins, and it is suggested that one or both of these analyses be used for screening palaeontological specimens. SIGNIFICANCE: This study has shown both chiral amino acid analysis and MALDI-ToF MS are reliable screening methods for predicting protein survival in fossils. Both these methods are quick, cheap, minimally destructive (1 mg and 15 mg respectively) and can provide crucial additional information about the endogeneity of the surviving proteins. It is hoped that the use of these screening methods will encourage the examination of a wide range of palaeontological specimens for potential proteomic analysis. This in turn will give us a better understanding of protein survival far back in time and under different environmental conditions.

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at proteome-wide scale and at peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 to identify cross-links induced in exponentially growing Bacillus subtilis cells treated in culture with bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of <5%. To also obtain a FDR <5% for non-redundant inter-protein cross-linked peptide pairs additional threshold values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions assigned to both composite peptides. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After application of the composite filter new protein-protein interactions were revealed among others between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur. SIGNIFICANCE: Important for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.

Breast cancer is the most common malignancy for women. Accurate prediction of breast cancer and its pathological stages is important for treatment decision-making. Although many studies have focused on discovering circulating biomarkers of breast cancer, no such biomarkers have been reported for different stages of this disease. In this study, we identified blood protein biomarkers for each stage of breast cancer by analyzing transcriptome and proteome data from patients. Analysis of the TCGA transcriptome datasets revealed that a large number of genes were differentially expressed in tumor samples of each stage of breast cancer compared with adjacent normal tissues. Blood-secretory proteins encoded by these genes were then predicted by bioinformatics programs. Furthermore, iTRAQ-based proteomic analysis was conducted for plasma samples of breast cancer patients with different stages. A portion of predicted blood-secretory proteins could be detected and verified differentially expressed. Finally, several proteins were chosen as potential blood protein biomarkers for different stages of breast cancer due to their consistent expression patterns at both mRNA and protein levels. Overall, our data provide new insights into diagnosis and classification of breast cancer as well as selection of optimal treatments. SIGNIFICANCE: We identified blood protein biomarkers for each stage of breast cancer by analyzing tissue-based transcriptome and blood-based proteome data from patients. To our knowledge, this is the first time to try to identify blood protein biomarkers for different stages of breast cancer via these integrative analyses. Our data may provide new insights into diagnosis and classification of breast cancer as well as selection of optimal treatment.

Filter-aided sample preparation (FASP) remains a popular choice for proteomic sample preparation, particularly for its ability to produce a 'clean' peptide sample clear of large molecule contaminants. However, sample loss continues to be a problem particularly for sample inputs that contain less than ten micrograms of protein. Here, we describe that the simple addition of a polymer, polyvinylpyrrolidone-40 (PVP-40) to the protein sample prior to FASP digest significantly improves peptide recovery and identifications, especially with lower level sample inputs. PVP-FASP produces clean samples which required no additional sample clean-up prior to nanoLC-MS analysis. In addition, PVP-FASP is compatible with other FASP modifications, including the use of sodium deoxycholate (DOC) to improve trypsin digestion. SIGNIFICANCE: Simple modification to FASP procedure improves sample recovery during proteomic digests in SDS, improving peptide identifications and median peptide intensity.

Equine recurrent uveitis (ERU) is a spontaneous, remitting-relapsing autoimmune disease driven by the adaptive immune system. Although T cells are described as the main effector cells in pathogenesis, granulocytes have also emerged as possible disease mediators. To explore the role of these innate immune cells, we investigated the whole cell proteome of granulocytes from equine recurrent uveitis cases and healthy controls. Among the 2362 proteins identified by mass spectrometry, we found 96 proteins with significantly changed abundance between groups (p < 0.05, fold change >1.2), representing 4.1% of total granulocyte proteome. Within these differential identifications, calgranulin B, a protein associated with pathogenesis in other autoimmune diseases, showed highest abundance in equine recurrent uveitis (18 fold). For a better interpretation of the results from our hypothesis-generating approach, we added a threshold for biological significance (ratio ERU/controls >2: 36 proteins) to the proteins with increased abundance in equine recurrent uveitis and analyzed their allocation to the subsets within the Immune System superpathway. The 36 differentially abundant proteins predominantly associated to RAF/MAP kinase cascade, MHC-I-mediated antigen presentation and neutrophil degranulation, suggesting a latently activated phenotype of these innate immune cells in disease. Raw data are available via ProteomeXchange with identifier PXD013648. SIGNIFICANCE: Our study provides new insights into the protein repertoire of primary equine granulocytes and identifies protein abundance changes associated to equine recurrent uveitis (ERU), an organ specific, spontaneously occurring autoimmune disease. We show that granulocyte proteins with increased abundance in ERU strongly associate to RAF/MAP kinase signaling, MHC-I antigen presentation and neutrophil degranulation, pointing to a more activated state of these cells in ERU cases. Since cells were obtained in quiescent stage of disease, latent activation of granulocytes underlines the role of these innate immune cells in ERU. These findings are highly relevant for veterinary medicine, further establishing the importance of granulocytes in this T cell-driven autoimmune disease. Moreover, they have translational quality for autoimmune uveitis in man, due to strong similarity in disease occurrence, progression and pathogenesis.

The small proteins and short open reading frames encoded peptides (SEPs) are of fundamental importance because of their essential roles in biological processes. However, the annotation or identification of them is challenging, in part owing to the limitation of the traditional genome annotation pipeline and their inherent characteristics of low abundance and low molecular weight. To discover and characterize SEPs in Hep3B cell line, we developed an optimized peptidomic assay by combining different peptide extraction and separation methods. The organic solvent precipitation method in peptidomic showed promotion in the enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high-quality MS/MS spectra. Furthermore, different strategies exhibited good complementarity in improving the total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Results in this work provide robust evidence to date that the human proteome is more complicated than previously appreciated, and this will be a benefit to discoveries of proteins without function annotation. SIGNIFICANCE: In this work, methods were optimized to identify SEPs in Hep3B. The organic solvent precipitation presents promotion in enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high quality MS/MS spectra. Different strategies exhibited good complementarity in improving total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Furthermore, 22 SEPs generated from the uORF may has potential effect in translation control, and 149 newly identified SEPs have known functional domains or cross-species conservation. Results in this work present robust evidence for the coding potential of the ignored region of human genomes and may provide additional insights into tumor biology.

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen causing meningitis in humans and pigs. However, information on the comparative protein expression of the blood-brain barrier (BBB) following SS2 infection is limited. Deletion of the serine/threonine kinase (stk) gene can decrease the ability of SS2 to invade the BBB. In the present study, bEnd.3 cells were used as the BBB model, and a SILAC comparative quantitative proteomic study of bEnd.3 cells infected with the SS2 ZY05719 or Δstk strain was performed to determine the differences between these strains infections. Compared with ZY05719-infected cells, 241 proteins were highly upregulated, and 81 were significantly downregulated in Δstk-infected cells. The obtained data revealed major changes in the proteins involved in RNA process, host cytoskeleton, tight junction disruption and immune response. Some differentially expressed proteins were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, and western blot analysis was used to validate the changes of some selected proteins at the translational level. The results obtained in this study may be useful to understand the host response to SS2 infection and provide crucial clues to decipher how STK expression in SS2 helps the bacteria penetrate the BBB. SIGNIFICANCE: A SILAC comparative quantitative proteomic assay was performed in bEnd.3 cells infected with the SS2 ZY05719 or Δstk strain. 241 upregulated and 81 downregulated differentially expressed proteins (DEPs) were identified. DEPs are involved in RNA process, host cytoskeleton, tight junction disruption and immune response. Some DEPs were examined by qPCR and western blot assays, which were similar to those of their corresponding proteins in the quantitative proteomics analysis.

Comparing multiple label-free shotgun proteomics datasets requires various data processing and formatting steps, including peptide-spectrum matching, protein inference, and quantification. Finally, the compilation of results files into a format that allows for downstream analyses. ProtyQuant performs protein inference and quantification calculations, and combines the results of individual datasets into plain text tables. These are lightweight, human-readable, and easy to import into databases or statistical software. ProtyQuant reads validated pepXML from proteomic workflows such as the Trans-Proteomic Pipeline (TPP), which makes it compatible with many commercial and free search engines. For protein inference and quantification, a modified version of the PIPQ program (He et al. 2016) was integrated. In contrast to simple spectral-counting, PIPQ sums up peptide probabilities. For assigning peptides to proteins, three algorithms are available: Multiple Counting, Equal Division, and Linear Programming. The accumulated peptide probabilities (app) are used for both tasks, protein probability estimation, and quantification. ProtyQuant was tested using a reference dataset for label-free shotgun proteomics, obtained from different concentrations of 48 human UPS proteins spiked into yeast lysate. Compared to ProteinProphet, ProtyQuant detected up to 126 (15%) more proteins in the mixture, applying an equal false positive rate (FPR). Using the app values for label-free quantification showed suitable sensitivity and linearity. Strikingly, the app values represent a realistic measure of 'Protein Presence,' an integral concept of protein probability and quantity. ProtyQuant provides a graphical user interface (GUI) and scripts for console-based processing. It is available (GNU GLP v3) for Windows, Linux, and Docker from https://bitbucket.org/lababi/protyquant/. SIGNIFICANCE: Integrating data from multiple shot-gun proteomics experiments overwhelms non-expert researchers. ProtyQuant complements well-established workflows by aiding the comparison of proteins across samples. Importantly, the probability and abundance of proteins are seen from a holistic point of view. The accumulated peptide probability (app) as an integral measure of 'Protein Presence' demonstrated reliable performance for both protein identification and quantification. Using the app as a single measure facilitates the compilation of reports in comparative proteomics.

The analysis of biological fluids to identify proteins that may indicate a disease setting, state and progression, is an increasingly explored field. Despite the expectatives created, there are several hurdles that must be solved to reach an extensive proteome coverage using mass spectrometry, mainly due to the complex composition of the matrices. In this regard, bile is specially challenging and yet, very attractive, as a proximal fluid that might provide valuable information for the management of liver and pancreas associated diseases. Proteins account for less than 5% of bile organic components and, although optimized protocols for protein extraction have been developed, only partial descriptions of bile proteome have been achieved. In this manuscript a new procedure is described that significantly improves protein recovery from rat bile, which reduces by a factor of six the sample amount required for a typical proteomics analysis. Moreover, the number of proteins reliably identified in a single nanoLC-MS/MS run from 1 μg protein was increased by three-fold. This procedure provides a valuable resource to dig deeper into the molecular composition of bile and open new avenues to identify new hallmarks of disease such as cholangiocarcinoma, hepatocellular carcinoma and pancreatic cancer for their better clinical management.

A number of studies have reported aberrant glycosylation in connection with malignancy. Our investigation further expands on this topic through the examination of N-glycans, which could be associated with the resistance of advanced stage, high-grade non-mucinous ovarian cancer to platinum/taxane based chemotherapy. We used tissue samples of 83 ovarian cancer patients, randomly divided into two independent cohorts (basic and validation). Both groups involved either cases with/without postoperative tumor residue or the cases determined either resistant or sensitive to this chemotherapy. In the validation cohort, preoperative serum samples were also available. N-glycans released from tumors and sera were permethylated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS analysis yielded a consecutive detection of 68 (tissue) and 63 (serum) N-glycan spectral signals. Eight of these were found to be differentially abundant in tissues of both independent cohorts including the cases with a postoperative cancer residue. One of these glycans was detected as differentially abundant in sera of the validation cohort. No statistically significant differences in intensities due to the same N-glycans were found in the cases without postoperative macroscopic residues in either the basic or validation cohort. From the biochemical point of view, the statistically significant N-glycans correspond to the structures carrying bisecting (terminal) GlcNAc residue and tetra-antennary structures with sialic acid and/or fucose residues. Among them, six tissue N-glycans could be considered potential markers connected with a resistance to chemotherapy in ovarian cancer patients. The prediction of primary resistance to standard chemotherapy may identify the group of patients suitable for alternative treatment strategies. SIGNIFICANCE: Drug resistance has become a major impediment to a successful treatment of patients with advanced ovarian cancer. The glycomic measurements related to cancer are becoming increasingly popular in identification of the key molecules as potential diagnostic and prognostic indicators. Our report deals with identification of differences in N-glycosylation of proteins in tissue and serum samples from the individuals showing sensitivity or resistance to platinum/taxane-based chemotherapy. The detection sensitivity to chemotherapy is vitally important for these patients.

The snail Bellamya purificata is an ecologically and economically important freshwater gastropod species. However, limited genomic resources are available for this snail. In this study, the transcriptome of mantle tissues and proteome of shells of B. purificata with two shell colors (namely light-cyan line (LC) and light-purple line (LP)) were deeply sequenced and characterized. A total of 5.72 million contigs were assembled into 157,015 unigenes, 21,455 (13.66%) of these unigenes were significantly matched to NR, Swiss-Prot, KOG, GO and KEGG database. 1807 differentially expressed genes (DEGs) were identified between the two different shell color lines. These DEGs were significantly enriched in five KEGG pathways including tyrosine metabolism, tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, and histidine metabolism, which suggested that the shell color polymorphism in B. purificata was a result of melanin synthesis variation. A total of 1521 proteins were identified in B. purificata here as well. The differentially expressed protein analysis showed that the tyrosinase content in LP was significantly decreased in comparison to LC, which agreed with the transcriptome analysis results. This study provides valuable genomic resources of B. purificata and improves our understanding of molecular mechanisms of biomineralization and shell color polymorphism in snail.

Short open reading frame-encoded peptides (SEP) represent a widely undiscovered part of the proteome. The detailed analysis of SEP has, despite inherent limitations such as incomplete sequence coverage, challenges encountered with protein inference, the identification of posttranslational modifications and the assignment of potential N- and C-terminal truncations, predominantly been assessed using bottom-up proteomic workflows. The use of top-down based proteomic workflows is capable of providing an unparalleled level of characterization information, which is of increased importance in the case of alternatively encoded protein products. However, top-down based analysis is not without its own limitations, for which efficient separation prior to MS analysis is a major issue. We established a sample preparation approach for the combined bottom-up and top-down proteomic analysis of SEP. Key improvements were made by the application of solid phase extraction (SPE), which supported enrichment of proteins below ca. 20 kDa, followed by 2D-LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterization of 36 proteoforms mapping to 12 SEP from the archaeon Methanosarcina mazei strain Gö1, with the concurrent detection and identification of several posttranslational modifications in SEP. BIOLOGICAL SIGNIFICANCE: Small or short open reading frames (sORF) have been widely neglected in genome research in the past. With their increasing discovery, the question about the presence and molecular function of their translation products, the short open reading frame-encoded peptides (SEP), arises. As these small proteins are usually below the 10 kDa range, the number of peptides identifiable by bottom-up proteomics is limited which hampers both the identification and the recognition of potential posttranslational modifications. The presented top-down approach allowed for the detection of full length SEP, as well as of terminally truncated proteoforms, and further enabled the identification of disulfide bonds in these small proteins. This demonstrates, that this yet widely undiscovered part of the proteome undergoes the same modifications as classical proteins which is an essential step for future understanding of the biological functions of these molecules.

The emerging technology of urinary proteomics has become an efficient biological approach for identifying biomarkers and characterizing pathogenesis in renal involvement. In this study, we attempted to elucidate the relationship between IgAN and HSPN in children, employing LC-MS/MS to perform urinary proteomic analyses using the DIA method. Early-morning spot urine was collected from patients with biopsy-proven IgAN (n = 19) and HSPN (n = 19) prior to treatment and renal biopsy in the Department of Pediatrics, Jinling Hospital, Nanjing, China, and did healthy volunteers (n = 14), from June 2018 to December 2019. Two hundred seventy-six urinary proteins and 125 urinary proteins were determined to be differentially expressed in children with IgAN (n = 4) and HSPN (n = 4), respectively, compared to the urinary proteins of healthy children (n = 4) (p < 0.05). GO analysis demonstrated that the differentially expressed proteins of the two groups, which were located in the extracellular matrix and cell membrane, were primarily involved in biological processes, including metabolic processes, immune system processes, cellular adhesion, cell proliferation, signaling, and biological regulation. KEGG analysis revealed that the differentially expressed proteins of the two groups were associated with cell adhesion molecules, ECM-receptor interactions, the PI3K-Akt signaling pathway, the complement and coagulation cascades, regulation of actin cytoskeleton, cholesterol metabolism, and platelet activation. The target proteins (alpha-1B-glycoprotein (A1BG) and afamin (AFM)), which participated in the complement and coagulation cascades and the regulation of complement activation, were further investigated in the independent validation cohort by ELISA. These proteins were significantly increased in children with IgAN (n = 15) and HSPN (n = 15) compared with the proteins observed in healthy controls (n = 10, P < 0.05). The validated results were consistent with the mass spectrometry results. SIGNIFICANCE: IgAN and HSPN both result from the glomerular deposition of abnormally glycosylated IgA1 with mesangial proliferative changes, and both diseases are common glomerulopathies in the pediatric population that are believed to be correlated. Interestingly, our data, by combining urinary proteomic analyses, showed that several uniform enrichment pathways played an important role in the progression of IgAN and HSPN, suggesting that we might reduce the renal involvement of the two diseases in children through these pathways. The same urinary proteins along these pathways were observed to be differentially expressed in children with IgAN and HSPN, implying that these proteins may be potential biomarkers to identify the two diseases. Future studies examining larger cohorts are warranted to confirm the validity of our findings.

Nephrotic syndrome is characterized by urinary excretion of plasma proteases or proteasuria. There is a lack of data on the quantity, activity status and identity of these aberrantly filtered proteases. We established a fluorescence-based substrate assay to quantify protease activity in urine samples from healthy and nephrotic humans and mice. Protease class activity was determined after addition of specific inhibitors. Individual proteases were identified by tandem mass spectrometry (MS/MS). In spot urine samples from 10 patients with acute nephrotic syndrome of various etiology, urinary protease activity was significantly increased compared to that of healthy persons (753 ± 178 vs. 244 ± 65 relative units, p < 0.05). The corresponding proteases were highly sensitive to inhibition by the serine protease inhibitors AEBSF (reduction by 85 ± 6% and 72 ± 8%, respectively) and aprotinin (83 ± 9% vs. 25 ± 6%, p < 0.05). MS/MS of all urinary proteins or after AEBSF purification showed that most of them were active serine proteases from the coagulation and complement cascade. These findings were recapitulated in mice, pointing to a similar pathophysiology. In conclusion, nephrotic syndrome leads to increased urinary excretion of active plasma proteases which can be termed proteasuria. Serine proteases account for the vast majority of urinary protease activity in health and nephrotic syndrome. SIGNIFICANCE STATEMENT: In this study, we found that nephrotic urine samples of humans and mice have a significantly increased protease activity compared to healthy urine samples, using a universal pentapeptide substrate library. This was driven by increased excretion of aprotinin-sensitive serine proteases. With tandem mass spectrometry, we provide a comprehensive and systematic overview of all urinary proteases or the "urine proteasome". We identified renally expressed proteases in health and addition of proteases from the coagulation and complement cascade in the nephrotic state. These results set the basis to study the role of urinary proteases at both health and nephrotic syndrome to find diagnostic markers of renal disease as well as possible therapeutic targets.

Fructus Rosae Roxburghii (FRR) has been considered as edible and medicinal fruit possessing antiatherosclerotic effect, but the mechanism is still unclear. HLP is material basis for AS formation. Under FRR action, TC, TG, LDL, HDL and ASI in serum were regulated to control level. Differentially expressed proteins in liver were analyzed by using TMT labeling and LC-MS/MS for better understanding the effect and molecular mechanism of FRR on diet-induced hyperlipidemic mice. In total, 4460 proteins were quantified, of which 469 proteins showed dramatic changes between each group. According to molecular functions, 25 differentially co-expressed proteins were divided into five categories: substance metabolism, energy transformation and signal transduction, transcription and translation, immune defense. 15 key proteins involved lipids metabolism, which were identified as Cyp7a1, Cyp3a11, Tm7sf2, COAT2, CSAD, RBP3, Lpin1, Dhrs4, Aldh1b1, GK, Acot 4, TSC22D1, PGFS, EHs, GSTM1. This suggested that FRR could maintain metabolic homeostasis by regulating the metabolism of fatty acids, biosynthesis of BAs and steroids, and production of LPOs. 20 oxidative lipids further confirmed their importance regulating lipids metabolism. It's first time potential antiatherosclerotic mechanism of FRR regulating blood lipids was explored from protein level, which is of great significance to explore new drug targets for AS. SIGNIFICANCE: Under the action of FRR juice, the blood lipids in mice were regulated to control level. By TMT proteomic analysis, the effect and molecular mechanism of FRR on diet-induced hyperlipidemic mice were further explored. 25 differentially co-expressed proteins obtained in three diet groups might cooperatively regulate the lipids metabolism and hepatic function of mice, thus maintaining the metabolism homeostasis. By lipidomics analysis, 20 oxidative lipids further confirmed the importance of ω-3 and ω-6 PUFAs in regulating the lipids metabolism. These findings provide an improved understanding for the regulation of FRR on the blood lipids and explores potential metabolic targets for AS prevention.

Protein ubiquitylation regulates almost all aspects of the biological processes including gene expression, DNA repair, cell proliferation and apoptosis in eukaryotic cells. Dysregulation of protein ubiquitylation caused by abnormal expression of enzymes in the ubiquitin system results in the onset of many diseases including cancer, neurodegenerative diseases, and metabolic syndromes. Therefore, targeting the ubiquitin system becomes a promising research area in drug discovery. Identification of protein ubiquitylation sites is critical for revealing the key ubiquitylation events associated with diseases and specific signaling pathways and for elucidating the biological functions of the specific ubiquitylation events. Many approaches that enrich for the ubiquitylated proteins and ubiquitylated peptides at the protein and peptide levels have been developed to facilitate their identification by MS. In this paper, we will review the proteomic approaches available for the identification of ubiquitylation events at the proteome scale and discuss their advantages and limitations. We will also brief the application of the profiling of ubiquitylation events in drug target discovery and in target validation for proteolysis-targeting chimera (PROTAC). Possible future research directions in this field will also be discussed. SIGNIFICANCE: Ubiquitylation plays critical roles in regulating many biological processes in eukaryotic cells. Identification of ubiquitylation sites can provide the essential information for the functional study of the specific modified substrates. Since ubiquitylated proteins have much lower abundance than non-ubiquitylated proteins, enrichment of ubiquitylated proteins or peptides is critical for their identification by MS. This review focuses on different enrichment approaches that facilitate their isolation and identification by MS and discusses the advantages and drawbacks of these approaches. The application of the profiling of ubiquitylation events in drug target discovery and future research directions will be beneficial to the research community.

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM. The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H2O2. κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins. Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SIGNIFICANCE: Donkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy. DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring. DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H2O2. In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits. Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population. Data of this study represent the largest inventory of proteins identified in Equidae milk, so far.

In the context of human evolution, the study of proteins may overcome the limitation of the high degradation of ancient DNA over time to provide biomolecular information useful for the phylogenetic reconstruction of hominid taxa. In this study, we used a shotgun proteomics approach to compare the tooth proteomes of extant human and non-human primates (gorilla, chimpanzee, orangutan and baboon) in order to search for a panel of peptides able to discriminate between taxa and further help reconstructing the evolutionary relationships of fossil primates. Among the 25 proteins shared by the five genera datasets, we found a combination of peptides with sequence variations allowing to differentiate the hominid taxa in the proteins AHSG, AMBN, APOA1, BGN, C9, COL11A2, COL22A1, COL3A1, DSPP, F2, LUM, OMD, PCOLCE and SERPINA1. The phylogenetic tree confirms the placement of the samples in the appropriate genus branches. Altogether, the results provide experimental evidence that a shotgun proteomics approach on dental tissue has the potential to detect taxonomic variation, which is promising for future investigations of uncharacterized and/or fossil hominid/hominin specimens. SIGNIFICANCE: A shotgun proteomics approach on human and non-human primate teeth allowed to identify peptides with taxonomic interest, highlighting the potential for future studies on hominid fossils.

This prospective study included four dog groups (group A: healthy dogs, groups B: dogs with idiopathic epilepsy under antiepileptic medication (AEM), C: idiopathic epilepsy dogs without AEM administration, D: dogs with structural epilepsy). The purpose of the study was to compare the proteomic profile among the four groups. Samples were analyzed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD018893. Eighteen proteins were statistically significant among the four groups (P < 0.05). MMP2 and EFEMP2 appeared down-regulated whereas HP and APO-A1 were up-regulated (groups B, D). CLEC3B and PEBP4 were up-regulated whereas APO-A1 was down-regulated (group C). IGLL1 was down-regulated (groups B, C) and up-regulated (group D). EFEMP2 was the only protein detected among the four groups and PEBP4 was significantly different among the epileptic dogs. Western blot and SPARCL immunoassay were used to quantify HP abundance change, validating proteomic analysis. Both, showed good correlation with HP levels identified through proteomic analysis (r = 0.712 and r = 0.703, respectively). SIGNIFICANCE: The proteomic analysis from CSF of dogs with epileptic seizures could reflect that MMP2, HP and APO-A1 may contribute to a blood-brain barrier disruption through the seizure-induced inflammatory process in the brain. MMP2 change may indicate the activation of protective mechanisms within the brain tissue. Antiepileptic medication could influence several cellular responses and alter the CSF proteome composition.

The selection of growth media is a very important consideration of any cell-based proteomics experiment. Alterations thereof may result in differences in basal proteomes simply due to disparities in the metabolite composition of the media. We investigate the effect of growth media on the proteomes of three microorganisms, specifically E. coli, S. cerevisiae, and S. pombe, using tandem mass tag (TMT)-based quantitative proteomics. We compared the protein abundance profiles of these microorganisms propagated in two distinct growth media that are commonly used for the respective organism. Our sample preparation strategy included SP3 bead-assisted protein isolation and digestion. In addition, we assembled a replicate set of samples in which we altered the proteolytic digestion from sequential treatment with LysC and trypsin to only LysC. Despite differences in peptides identified and a drop in quantified proteins, the results were similar between the two datasets for all three microorganisms. Approximately 10% of the proteins of each respective microorganism were significantly altered in each dataset. As expected, gene ontology analysis revealed that the majority of differentially expressed proteins are implicated in metabolism. These data emphasize further the importance and the potential consequences of growth media selection. SIGNIFICANCE: Various microorganisms are used as model systems throughout in biological studies, including proteomics-based investigations. The growth conditions of these organisms are of utmost importance, of which one major consideration is the choice of growth media. We hypothesize that growth media selection has a considerable impact on the baseline proteome of a given microorganism. To test this hypothesis, we used tandem mass tag (TMT)-based quantitative multiplexed proteomics to profile the proteomes of E. coli, S. cerevisiae, and S. pombe each grown in two different, yet common, growth media for the respective species. Our data show that approximately 10% of the proteins of each respective microorganism were significantly altered and that many of the differentially expressed proteins are implicated in metabolism. We provide several datasets which are potentially valuable for growth media selection with respect to downstream biochemical analysis.

Salinity can induce Mesembryanthemum crystallinum to shift its photosynthesis from C3 to crassulacean acid metabolism (CAM), leading to enhanced plant water use efficiency. Studying how M. crystallinum changes its carbon fixation pathways is important for potential translation into crops and enhancing crop resilience. In this study, we examined proteomic changes in guard cells and mesophyll cells in the course of the C3 to CAM transition. We collected enriched guard cells and mesophyll cells during a short period of transition. A total of 1153 proteins were identified and quantified in the two cell-types. During the transition, proteins in the guard cells and mesophyll cells exhibited differential changes. For example, we observed nocturnal carbon fixation in mesophyll cells and proteins involved in cell growth in the two cell-types. Proteins involved in osmotic adjustment, ion transport, energy metabolism and light response may play important roles in the C3 to CAM transition. Real-time PCR experiments were conducted to determine potential correlations between transcript and protein levels. These results have highlighted potential molecular mechanisms underlying the C3 to CAM transition of guard cells and mesophyll cells of the important facultative CAM plant. BIOLOGICAL SIGNIFICANCE: Fresh water resource for agricultural food production is a global challenge. Nature has evolved crassulacean acid metabolism (CAM) plants with enhanced water use efficiency. Using single cell-type proteomics, this study revealed molecular changes taking place in guard cells and mesophyll cells during the shift of ice plant photosynthesis from C3 to CAM. The results have provided important insights into the CAM transition and may facilitate effort toward enhancing crop resilience for global food security.

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and β-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SIGNIFICANCE: Previously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins. : Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome.

The long-term established symbiosis between gut microbiota and humans is based upon a dynamic equilibrium that, if unbalanced, could lead to the development of diseases. Despite the huge amount of data concerning the microbiota-gut-brain-axis, little information is available on what happens at the molecular level in bacteria, when exposed to human signals. In the present study, the physiological effects exerted by norepinephrine (NE), a human hormone present in significant amounts in the host gut, were analyzed using the commensal/probiotic strain Enterococcus faecium NCIMB10415 as a target. The aim was to compare the protein profiles of treated and untreated bacteria and relating these proteome patterns to some phenotypic modifications important for bacteria-host interaction. Actually, to date, only pathogens have been considered. Combining a gel-free/label-free proteomic analysis with the evaluation of bile salts resistance, biofilm formation and autoaggregation ability (as well as with the bacterial growth kinetics), allowed to detect changes induced by NE treatment on all the tested probiotic properties. Furthermore, exposure to the bioactive molecule increased the abundance of proteins related to stress response and to host-microbe interaction, such as moonlight proteins involved in adhesion and immune stimulation. The results of this investigation demonstrated that, not only pathogens, but also commensal gut bacteria are affected by host-derived hormones, underlining the importance of a correct cross-signalling in the maintenance of gut homeostasis. SIGNIFICANCE: The crucial role played by the human gut microbiota in ensuring host homeostasis and health is definitively ascertained as suggested by the holobiome concept. The present research was intended to shed light on the endocrinological perturbations possibly affecting microbiota. The microbial model used in this study belongs to Enterococcus faecium species, whose controversial role as gut commensal and opportunistic pathogen in the gut ecosystem is well recognized. The results obtained in the present investigation clearly demonstrate that E. faecium NCIMB10415 can sense and respond to norepinephrine, a human hormone abundant at the gut level, by changing protein profiles and physiology, inducing changes that could favor survival and colonization of the host tissues. To our knowledge, this is the first proteomic report concerning the impact of a human hormone on a commensal/probiotic bacterium, since previous research has focused on exploring the effects of neuroendocrine molecules on growth and virulence of pathogenic species.

Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20-300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SIGNIFICANCE: This study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.

Microgravity induces injury of intestinal barrier. However, the underlying mechanism remains unclear. The present study aimed to investigate the pathological change of intestinal mucosa induced by long term simulated microgravity and to explore its etiological mechanism using a proteomic approach. The well accepted tail-suspended rat model was used to simulate microgravity. The damage of rat small intestine was evaluated via histological and molecular test, and a label-free comparative proteomic strategy was used to determine the molecular mechanism. Simulated microgravity for 21 days damaged intestine barrier with decreased numbers of the goblet cells, large intercellular space, and down-regulated adhesion molecules, accompanied by increased intestinal permeability. Proteomic analysis identified 416 differentially expressed proteins and showed simulated microgravity dramatically down-regulated the adhesion molecules and deteriorated several pathways for metabolism, focal adhesion, and regulation of actin cytoskeleton. Western-blot analysis confirmed that myosin regulatory light chain (MLC) 12B was significantly down-regulated, while rho-associated protein kinase, myosin light chain kinase (MLCK), and phosphorylated MLC were dramatically up-regulated. Taken together, these data reveal that down-regulation of adhesion molecules and MLCK dependent up-regulation MLC phosphorylation mediate intestinal barrier dysfunction during simulated microgravity injury. Our results also indicate that regulation of epithelial MLCK is a potential target for the therapeutic treatment of microgravity injury.

The detection of disease-related plasma biomarkers has challenged the proteomic community for years. Attractive features for plasma proteomics includes the ease of collection and small volume needed for analysis, but on the other hand, the presence of highly abundant proteins complicates sample preparation procedures and reduces dynamic range. Data independent acquisition label free quantitation (DIA-LFQ) by mass spectrometry partly overcomes the dynamic range issue; however, generating the peptide spectral reference libraries that allow extensive analysis of the plasma proteome can be a slow and expensive task which is unattainable for many laboratories. We investigated the re-purposing of publically available plasma proteome datasets and the impact on peptide/protein detection for DIA-LFQ. We carried out these studies in the context of identifying putative biomarkers of response to neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma, as no useful plasma biomarkers have been clinically adopted. We demonstrated the benefit in searching DIA data against multiple spectral libraries to show that complement proteins were linked to NAC response in PDAC patients, confirming previous observations of the prognostic utility of complement following adjuvant chemotherapy. Our workflow demonstrates that DIA-LFQ can be readily applied in the oncology setting for the putative assignment of clinically relevant plasma biomarkers. STATEMENT OF SIGNIFICANCE: The proteomic mass spectrometry analysis of undepleted, unfractionated human plasma has benefits for sample throughput but remains challenging to obtain deep coverage. This work evaluated the re-purposing of open source peptide mass spectrometry data from human plasma to create spectral reference libraries for use in Data independent acquisition (DIA). We showed how seeding in locally acquired data to integrate iRT peptides into spectral libraries increased identification confidence by facilitating querying of multiple libraries. This workflow was applied to the discovery of putative plasma biomarkers for response to neoadjuvant chemotherapy (NAC) in pancreatic ductal adenocarcinoma patients. There is a paucity of prior information in the literature on this topic and we show that good responder patients have reduced levels of complement proteins.

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Successful Plasmodium falciparum invasion of red blood cells includes the orderly execution of highly specific receptor-ligand molecular interactions between the parasite's proteins and the red blood cell membrane proteins. There is a growing need for elucidating receptor-ligand pairings, which will help in understanding the parasite's biology and provide the fundamental basis for developing prophylactic or therapeutic alternatives leading to mitigating or eliminating this type of malaria. We have thus used Plasmodium falciparum RH5 - derived peptides and ghost red blood cell proteins in synthetic peptide affinity capture assays to identify important host receptors used by Plasmodium spp. in the invasion of red blood cells. LC-MS/MS analysis confirmed the extensively described interaction between PfRH5 and the basigin receptor on the red blood cell membrane. As shown here, tagged synthetic peptides displaying high binding ability to erythrocytes can be used to identify receptors present in protein extracts from ghost red blood cells via affinity capture and LC-MS/MS. SIGNIFICANCE: The article describes a novel approach for identifying red blood cell receptors based on the ability of synthetic peptides having high red blood cell binding capacity to capture Plasmodium spp. receptors on proteins extracted from ghost red blood cells. Specifically, novel methods to identify Plasmodium falciparum reticulocyte binding protein homolog 5 PfRH5 and basigin interaction using a combination of affinity capture and LC-MS/MS assays is described. Identification of these host RBC receptors interacting with malarial parasite proteins is of utmost importance in studying the disease's pathogenesis and will provide crucial information in understanding the parasite's biology. In addition, data from these studies can be used to identify potential therapeutic target(s) to mitigate or eliminate this debilitating disease.

Late diagnosis of pancreatic ductal adenocarcinoma (PDA) is one of the reasons of its low 5-year survival rate and it is due to its unspecific symptoms during the first stages of the disease and the lack of reliable serological markers. Since PDA shows an altered glycan expression, here we have focused on finding novel potential biomarkers, namely glycoproteins that express the tumor associated carbohydrate structure sialyl-Lewis x (sLex), which is described in PDA. Through a glycoproteomic approach, we have analyzed target proteins containing sLex from PDA tissues by 2DE and immunodetection techniques, and have identified by mass spectrometry the protein MFAP4 as a carrier of sLex in PDA. MFAP4 showed a higher expression in PDA tissues compared with pancreatic control tissues. In addition, the colocalization of sLex over MFAP4 was found only in PDA and not in control pancreatic tissues. The analysis of MFAP4 expression in PDA cell lines and their secretome, in combination with immunohistochemistry of pancreatic tissues, revealed that MFAP4 was not produced by PDA cells, but it was found in the pancreatic extracellular matrix. The specificity of MFAP4 glycoform containing sLex in PDA tissues shows its relevance as a potential PDA biomarker. SIGNIFICANCE: Despite advances in the field of cancer research, pancreatic ductal adenocarcinoma (PDA) lacks of a specific and sensitive biomarker for its early detection, when curative resection is still possible before metastases arise. Thus, efforts to discover new PDA biomarkers represent the first line in the fight against the increase of its incidence reported in recent years. Glycan alterations on glycoconjugates, such as glycoproteins have emerged as a rich source for the identification of novel cancer markers. In the present work, we aimed to shed light on novel biomarkers based on altered glycosylation in PDA, in particular those glycoproteins of PDA tissues carrying the tumor carbohydrate antigen sialyl-Lewis x (sLex). Through a glycoproteomic approach, we have shown that the glycoprotein MFAP4 carries sLex in PDA tissues and not in control pancreatic tissues. MFAP4 is found in the extracellular matrix in PDA and although its role in cancer progression is unclear, its sLex glycoform could be a potential biomarker in pancreatic ductal adenocarcinoma.

The proteomic analysis of hairs, yarns or textiles has emerged as a powerful method to determine species of origin, mainly used in archaeozoological research and fraud control. Differentiation between the South American camelid (SAC) species (the wild guanaco and vicuña and their respective domesticates the llama and alpaca) is particularly challenging due to poor database information and significant hybridization between species. In this study, we analysed 41 modern and 4 archaeological samples from the four SACs species. Despite strong similarities with Old World Camelidae, we identified 7 peptides specific to SACs assigned to keratin K86 and the keratin-associated proteins KAP13-1 and KAP11-1. Untargeted multivariate analysis of the LC-MS data permitted to distinguish SAC species and propose discriminant features. MS/MS-based molecular networking combined with database-assisted de novo sequencing permitted to identify 5 new taxonomic peptides assigned to K33a, K81 and/or K83 keratins and KAP19-1. These peptides differentiate the two wild species, guanaco and vicuña. These results show the value of combining database search and untargeted metabolomic approaches for paleoproteomics, and reveal for the first time the potential of molecular networks to highlight deamidation related to diagenesis and cluster highly similar peptides related to interchain homologies or intra- or inter-specific polymorphism. SIGNIFICANCE: This study used an innovative approach combining multivariate analysis of LC-MS data together with molecular networking and database-assisted de novo sequencing to identify taxonomic peptides in palaeoproteomics. It constitutes the first attempt to differentiate between hair fibres from the four South American camelids (SACs) based on proteomic analysis of modern and archaeological samples. It provides different proteomic signatures for each of the four SAC species and proposes new SAC taxonomic peptides of interest in archaeozoology and fraud control. SACs have been extensively exploited since human colonization of South America but have not been studied to the extent of their economic, cultural and heritage importance. Applied to the analysis of ancient Andean textiles, our results should permit a better understanding of cultural and pastoral practices in South America. The wild SACs are endangered by poaching and black-market sale of their fibre. For the first time, our results provide discriminant features for the determination of species of origin of contraband fibre.

Concatenated target-decoy database searches are commonly used in proteogenomic research for variant peptide identification. Currently, protein-based and peptide-based sequence databases are applied to store variant sequences for database searches. The protein-based database records a full-length wild-type protein sequence but using the given variant events to replace the original amino acids, whereas the peptide-based database retains only the in silico digested peptides containing the variants. However, the performance of applying various decoy generation methods on the peptide-based variant sequence database is still unclear, compared to the protein-based database. In this paper, we conduct a thorough comparison on target-decoy databases constructed by the above two types of databases coupled with various decoy generation methods for proteogenomic analyses. The results show that for the protein-based variant sequence database, using the reverse or the pseudo reverse method achieves similar performance for variant peptide identification. Furthermore, for the peptide-based database, the pseudo reverse method is more suitable than the widely used reverse method, as shown by identifying 6% more variant PSMs in a HEK293 cell line data set. SIGNIFICANCE: In our survey of publications on proteogenomic studies, 57% of the studies adopt the peptide-based variant sequence database coupled with the reverse method for decoy generation to construct a target-decoy database for searches. However, our results show that when using the peptide-based variant sequence database, it is better to adopt the pseudo reverse method for generating decoy sequences, to avoid leading to fewer variant peptides being identified.

Identification and characterization of ancient proteins still require technical developments towards non-invasiveness, sensitivity, versatility and ease of use of the analyses. We report that the enzyme functionalized films, described in Cicatiello et al. (2018), can be used efficiently on the surface of different objects ranging from fixative-coated paper to canvas to the coating on an albumen photograph, as well as the much harder surfaces of ivory objects and the proteinaceous binders in the decoration of a wooden Egyptian coffin. The mixture of digested peptides that are efficiently captured on the functionalized surface are also amenable to LC-MS/MS analysis, which is necessary to confidently identify chemical modifications induced upon degradation, in order to characterize the conservation state of proteins. Moreover, in a two-step procedure, we have combined the trypsin functionalized film with a PNGaseF functionalized film, which adds a deglycosylation pretreatment allowing improved detection of glycosylated proteins. SIGNIFICANCE: User friendly trypsin functionalized films were implemented to expand their potential as versatile, modular tools that can be widely exploited in the world of diagnosis of cultural heritage objects, ancient proteins, and palaeoproteomics: a procedure that could be carried out by conservators or archaeologists first on-site and later analysed with standard MS techniques.

In order to optimize sample preparation for shotgun proteomics, we compared four cysteine alkylating agents: iodoacetamide, chloroacetamide, 4-vinylpyridine and methyl methanethiosulfonate, and estimated their effects on the results of proteome analysis. Because alkylation may result in methionine modification in vitro, proteomics data were searched for methionine to isothreonine conversions, which may mimic genomic methionine to threonine substitutions found in proteogenomic analyses. We found that chloroacetamide was superior to the other reagents in terms of the number of identified peptides and undesirable off-site reactions. Among the reagents evaluated, iodoacetamide increased the rate of methionine-to-isothreonine conversion, especially if the sample was prepared in gel. The presence of proline following methionine in a protein sequence increased the modification rate as well. Generally, the methionine-to-isothreonine conversion events were relatively rare, but should be taken into account in proteogenomic studies when searching for single nucleotide polymorphism events at the protein level. Additionally, we have evaluated other methionine modifications, such as oxidation and carbamidomethylation. We found that carbamidomethylation may affect up to 80% of peptides containing methionine under the condition of iodoacetamide alkylation. In this case, carbamidomethylation of methionine is more common than oxidation and should be accounted for as a variable modification during proteomic search. SIGNIFICANCE: One of the most trending questions in bottom-up proteomics is the depth of proteome profiling, in other words, the coverage of proteins by identified tryptic peptides. In proteogenomics, where the identification of a single peptide, e.g. bearing an amino acid substitution, may be of interest, high sequence coverage is especially important. Chemical modifications during sample preparation may mimic biologically significant coding mutations at the proteome level. A typical example of such modification is methionine to isothreonine conversion during alkylation, which mimics methionine to threonine substitution in protein sequences due to respective genomic mutations. Therefore, the studies on the proper selection of alkylating reagents which balance the cysteine alkylation efficiency and the extent of methionine conversion upon conventional proteomic sample preparation workflow are crucial for the outcome of proteogenomic analyses and should present a general interest for the proteomic community.

Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and impaired myocardial function. Endomyocardial biopsies (EMB) enable immunohistochemical and molecular characterization of this disease. However, knowledge about specific molecular patterns and their relation to cardiac function in both ventricles is rare. Therefore, we performed a mass spectrometric analysis of 28 paired EMBs of left (LV) and right ventricles (RV) of patients with DCM or suspected myocarditis allowing quantitative profiling of 743 proteins. We analysed associations between protein abundance of LV and RV as well as the echocardiographic parameters LVEF, TAPSE, LVEDDI, and RVEDDI by linear regression models. Overall, more LV than RV proteins were associated with LV parameters or with RVEDDI. Most LV and RV proteins increasing in level with impairing of LVEF were annotated to structural components of cardiac tissue. Additionally, a high proportion of LV proteins with metabolic functions decreased in level with decreasing LVEF. Results were validated with LV heart sections of a genetic murine heart failure model. The study shows, that remodelling and systolic dysfunction in DCM is mirrored by distinct alterations in protein composition of both ventricles. Loss of LV systolic function is reflected predominantly by alterations in proteins assigned to metabolic functions in the LV whereas structural remodelling was more obvious in the RV. Alterations related to intermediate filaments were seen in both ventricles and highlight such proteins as early indicators of LV loss of function. SIGNIFICANCE: The present study report protein sets in the RV and the LV being associated with ventricular function and remodelling in DCM. Protein abundances in the LV and the RV emphasize and expand current knowledge on pathophysiological changes in heart failure and DCM. While RV and LV EMBs do not differ concerning diagnostic assessment of inflammatory status and virus persistence, additional information reflecting disease severity associated protein alterations can be gained by EMB protein profiling. RV and LV protein data provided complementary information. The protein pattern of the LV reflects metabolic changes and an impaired energy production, which is associated with the degree of LV systolic dysfunction and remodelling and may yield important information about the disease status in DCM. On the other hand, at this disease stage of DCM with still preserved RV function, RV alterations in structural proteins may reflect myocardial compensatory protective mechanisms for maintenance of structure and cellular function. The study highlight particular proteins being of interest as heart failure biomarkers in both ventricles which seem to reflect the severity of the disease. Further comparative studies between different HF aetiologies have to evaluate those proteins as markers specific for DCM.

Multiple Myeloma (MM), the second most common lymphoid cancer worldwide, is characterised by the uninhibited proliferation of terminally differentiated B-lymphocytes. Leading to The diagnosis typically requires the presence of a monoclonal protein (M protein) and the demonstration of CRAB features (hypercalcemia, renal impairment, anaemia and bone lesions). MM is considered incurable as, due to serial clonal evolution, the vast majority of patients succumb to treatment-refractory disease. MGUS (Monoclonal Gammopathy of Unknown Uncertain Significance) is the pre-malignant form of MM and, although 93% of MM patients exhibit M protein production associated with MGUS before diagnosis, little is known about the switch from pre-malignant to malignant disease. To explore this disease transition further, LC-MS/MS analysis was carried out to identify potential salivary biomarkers to monitor disease burden. FABP5 was detected in saliva as having a significant increase in abundance when MGUS was compared to symptomatic MM. The levels of FABP5 decreased after treatment indicating correlation with tumour burden. This finding was validated using western blot analysis and ELISA analysis. SIGNIFICANCE: The field of biomarker discovery has focused largely on serum as a biofluid. Saliva is a readily available biofluid that, as a biomarker resource, has been relatively un-explored. The identification of changes in saliva indicating disease progression underlines the utility of saliva as a non-invasive source of informative biomarkers reflecting disease burden and progression.

Diarrheal irritable bowel syndrome (IBS-D) is a chronic functional bowel disease with no clear diagnostic markers and no satisfactory treatment strategies. In recent years, the importance of intestinal microstructure and function in IBS-D has been emphasized. However, the intestinal tissue proteomics of IBS-D patients has not been analyzed. Here, we systematically analyzed the molecule profiling of the intestinal tissues in IBS-D patients through tandem mass tag (TMT)-based proteomics for the first time, aiming to reveal the pathogenesis and provide evidence for diagnosis and treatment of IBS-D. Five IBS-D patients and five healthy subjects were selected, biopsy tissue samples from the junction of sigmoid and rectum were analyzed by TMT proteomics. Differentially expressed proteins were obtained and bioinformatics analysis was performed. Furthermore, parallel reaction monitoring (PRM) and q-PCR detection were applied to validate the differentially expressed proteins. Eighty differentially expressed proteins were screened, 48 of which were up-regulated and 32 were down-regulated (fold change >1.2, P < 0.05). Bioinformatics analysis showed that these proteins were significantly enriched in the nutrient ingestion pathways which are related to immune molecules. SELENBP1, VSIG2, HMGB1, DHCR7, BCAP31 and other molecules were significantly changed. Our study revealed the underlying mechanisms of IBS-D intestinal dysfunction. SIGNIFICANCE: Irritable bowel syndrome with diarrhea (IBS-D) is a worldwide chronic intestinal disease with no definite diagnostic markers. It is still a challenge to accurately locate the pathogenesis of patients for appropriate treatment strategy. Established proteomics studies of IBS-D are only based on urine, blood, or tissue samples from animals. Our study was the first TMT proteomics analysis on intestinal biopsy tissues of patients with IBS-D, which revealed the changes of molecular spectrum of actual intestinal conditions in patients with IBS-D. Some important molecules and signaling pathways have been found abnormal in our study, which were related with nutrient uptake. They not only provided preliminary clues for low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) intolerance, an unsolved conundrum of IBS-D, but also revealed obscure problems of protein, lipid, and other nutrients ingestion in IBS-D patients. Some of these differentially expressed molecules have been preliminarily verified, and will may be potential candidate molecules for diagnostic markers of IBS-D.

Quantifying spawning biomass of commercially relevant fish species is important to generate fishing quotas. This will mostly rely on the annual or daily production of fish eggs. However, these have to be identified precisely to species level to obtain a reliable estimate of offspring production of the different species. Because morphological identification can be very difficult, recent developments are heading towards application of molecular tools. Methods such as COI barcoding have long handling times and cause high costs for single specimen identifications. In order to test MALDI-TOF MS, a rapid and cost-effective alternative for species identification, we identified fish eggs using COI barcoding and used the same specimens to set up a MALDI-TOF MS reference library. This library, constructed from two different MALDI-TOF MS instruments, was then used to identify unknown eggs from a different sampling occasion. By using a line of evidence from hierarchical clustering and different supervised identification approaches we obtained concordant species identifications for 97.5% of the unknown fish eggs, proving MALDI-TOF MS a good tool for rapid species level identification of fish eggs. At the same time we point out the necessity of adjusting identification scores of supervised methods for identification to optimize identification success. SIGNIFICANCE: Fish products are commercially highly important and many societies rely on them as a major food resource. Over many decades stocks of various relevant fish species have been reduced due to unregulated overfishing. Nowadays, to avoid overfishing and threatening of important fish species, fish stocks are regularly monitored. One component of this monitoring is the monitoring of spawning stock sizes. Whereas this is highly dependent on correct species identification of fish eggs, morphological identification is difficult because of lack of morphological features.

Dental stem cells have great potential in clinical practice as an adult mesenchymal stem cell, such as dental follicle and the apical papilla, have strong proliferation and differentiation characteristics. The developmental relevance and discrimination of them in the niche is not clear, which limits their application scenarios. The aim of this study was to investigate the intrinsical differences in cellular contents of DFSCs and SCAP by Tandem mass tag (TMT) labeling quantitative proteomics. Cell lysates were labeled and tracked by the combined use of TMT and LC-MS/MS. A total of 1622 proteins were detected, of which 421 were different and 12 were significantly up-regulated and 4 were significantly down-regulated. The results of proteomics support the application of stem cells in the treatment of neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, Parkinson's disease and so on. The difference is related to cell proliferation and protection of neurons from inflammation and autophagy damage. Highly expressed proteins predict the special ability of DFSCs to stably proliferate and differentiate through CD13, MARCKS, and PAST1. The strong immune stability of SCAP is supported by NPC1.This study expands our understanding on the molecular mechanisms of tooth development and regeneration, and provide basic support for dental stem cells in clinical applications such as neurological and immune diseases.

Erratum in J Proteomics. 2021 Apr 30;238:104165.