Protein quantification strategies using multiple proteases have been shown to deliver poor interprotease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, toward 10 proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned 3 orders of magnitude (0.02-70 μM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.

Neuropeptides are signaling molecules that regulate almost all physiological processes in animals. Around 50 different genes for neuropeptides have been described in insects. In Coleoptera, which is the largest insect order based on numbers of described species, knowledge about neuropeptides and protein hormones is still limited to a few species. Here, we analyze the neuropeptidomes of two closely related tenebrionid beetles: Tenebrio molitor and Zophobas atratus─both of which are model species in physiological and pharmacological research. We combined transcriptomic and mass spectrometry analyses of the central nervous system to identify neuropeptides and neuropeptide-like and protein hormones. Several precursors were identified in T. molitor and Z. atratus, of which 50 and 40, respectively, were confirmed by mass spectrometry. This study provides the basis for further functional studies of neuropeptides as well as for the design of environmentally friendly and species-specific peptidomimetics to be used as biopesticides. Furthermore, since T. molitor has become accepted by the European Food Safety Authority as a novel food, a deeper knowledge of the neuropeptidome of this species will prove useful for optimizing production programs at an industrial scale.

Robust, efficient, and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here, we present a comprehensive comparison of the 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, and commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependency, and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project.

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of >200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

It is generally believed that vascular endothelial cells (VECs) rely on glycolysis instead of the tricarboxylic acid (TCA) cycle under both normoxic and hypoxic conditions. However, the metabolic pattern of human umbilical vein endothelial cells (HUVECs) under extreme ischemia (hypoxia and nutrient deprivation) needs to be elucidated. We initiated a lethal ischemic model of HUVECs, performed proteomics and bioinformatics, and verified the metabolic pattern shift of HUVECs. Ischemic HUVECs displayed extensive aerobic respiration, including upregulation of the TCA cycle and mitochondrial respiratory chain in mitochondria and downregulation of glycolysis in cytoplasm. The TCA cycle was enhanced while the cell viability was decreased through the citrate synthase pathway when substrates of the TCA cycle (acetate and/or pyruvate) were added and vice versa when inhibitors of the TCA cycle (palmitoyl-CoA and/or avidin) were applied. The inconsistency of the TCA cycle level and cell viability suggested that the extensive TCA cycle can keep cells alive yet generate toxic substances that reduce cell viability. The data revealed that HUVECs depend on "ischemic TCA cycle" instead of glycolysis to keep cells alive under lethal ischemic conditions, but consideration must be given to relieve cell injury.

The combination of ion mobility mass spectrometry (IM-MS) and chromatography is a valuable tool for identifying compounds in natural products. In this study, using an ultra-performance liquid chromatography system coupled to a high-resolution quadrupole/traveling wave ion mobility spectrometry/time-of-flight MS (UPLC-TWIMS-QTOF), we have established and validated a comprehensive TWCCSN2 and MS database for 112 plant specialized metabolites. The database included 15 compounds that were isolated and purified in-house and are not commercially available. We obtained accurate m/z, retention times, fragment ions, and TWIMS-derived CCS (TWCCSN2) values for 207 adducts (ESI+ and ESI-). The database included novel 158 TWCCSN2 values from 79 specialized metabolites. In the presence of plant matrix, the CCS measurement was reproducible and robust. Finally, we demonstrated the application of the database to extend the metabolite coverage of Ventilago harmandiana Pierre. In addition to pyranonaphthoquinones, a group of known specialized metabolites in V. harmandiana, we identified flavonoids, xanthone, naphthofuran, and protocatechuic acid for the first time through targeted analysis. Interestingly, further investigation using IM-MS of unknown features suggested the presence of organonitrogen compounds and lipid and lipid-like molecules, which is also reported for the first time. Data are available on the MassIVE (https://massive.ucsd.edu, data set identifier MSV000090213).

This study demonstrates how the latest ultrahigh-performance liquid chromatography (UHPLC) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nanoflow liquid chromatography-mass spectrometry (nanoLC-MS) methods. The increased back pressures from the UHPLC system enabled the use of 75 μm I.D. × 75 cm columns packed with 2 μm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time, which maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contributes to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with a deep dive into the protein level alterations. Data are available via ProteomeXchange with identifier PXD035665.