Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

Extracellular vesicles from plasma, other body fluids and cell culture media hold great promise in the search for biomarkers. Exosomes in particular, the vesicle type that is secreted after being produced in the endocytic pathway and having a diameter of 30-150 nm, are considered to be a conveyance for signaling molecules and, therefore, to hold valuable information regarding the health and activity status of the cells from which they are released. The vesicular nature of exosomes is central to all methods used to separate them from the highly abundant proteins in plasma and other fluids. The enrichment of the vesicles is essential for mass spectrometry-based analysis as they represent only a very small component of all plasma proteins. The progression of isolation techniques for exosomes from ultracentrifugation through chromatographic separation using hydrophobic packing materials shows that effective enrichment is possible and that high throughput approaches to exosome enrichment are achievable.

Due to their involvement in numerous biochemical pathways, neuropeptides have been the focus of many recent research studies. Unfortunately, classic analytical methods, such as western blots and enzyme-linked immunosorbent assays, are extremely limited in terms of global investigations, leading researchers to search for more advanced techniques capable of probing the entire neuropeptidome of an organism. With recent technological advances, mass spectrometry (MS) has provided methodology to gain global knowledge of a neuropeptidome on a spatial, temporal, and quantitative level. This review will cover key considerations for the analysis of neuropeptides by MS, including sample preparation strategies, instrumental advances for identification, structural characterization, and imaging; insightful functional studies; and newly developed absolute and relative quantitation strategies. While many discoveries have been made with MS, the methodology is still in its infancy. Many of the current challenges and areas that need development will also be highlighted in this review.

Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels. Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system-wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)-based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically. This review focuses on the investigation of extracellular glycoproteins using MS-based proteomics. We first discuss the methods for selectively enriching surface glycoproteins and investigating protein interactions on the cell surface, followed by the application of MS-based proteomics for surface glycoprotein dynamics analysis and biomarker discovery. We then summarize the methods to comprehensively study secreted glycoproteins by integrating various enrichment approaches with MS-based proteomics and their applications for global analysis of secreted glycoproteins in different biological samples. Collectively, MS significantly expands our knowledge of extracellular glycoproteins and enables us to identify extracellular glycoproteins as potential biomarkers for disease detection and drug targets for disease treatment.

Exosomes (small extracellular vesicles) in living organisms play an important role in processes such as cell proliferation or intercellular communication. Recently, exosomes have been extensively investigated for biomarker discoveries for various diseases. An important aspect of exosome analysis involves the development of enrichment methods that have been introduced for successful isolation of exosomes. These methods include ultracentrifugation, size exclusion chromatography, polyethylene glycol-based precipitation, immunoaffinity-based enrichment, ultrafiltration, and asymmetric flow field-flow fractionation among others. To confirm the presence of exosomes, various characterization methods have been utilized such as Western blot analysis, atomic force microscopy, electron microscopy, optical methods, zeta potential, visual inspection, and mass spectrometry. Recent advances in high-resolution separations, high-performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Herein we review various exosome enrichment methods, characterization methods, and recent trends of exosome investigations using mass spectrometry-based approaches for biomarker discovery.

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is one of the least specimen destructive ambient ionization mass spectrometry tissue imaging methods. It enables rapid simultaneous mapping, measurement, and identification of hundreds of molecules from an unmodified tissue sample. Over the years, since its first introduction as an imaging technique in 2005, DESI-MSI has been extensively developed as a tool for separating tissue regions of various histopathologic classes for diagnostic applications. Recently, DESI-MSI has also emerged as a versatile technique that enables drug discovery and can guide the efficient development of drug delivery systems. For example, it has been increasingly employed for uncovering unique patterns of in vivo drug distribution, the discovery of potentially treatable biochemical pathways, revealing novel druggable targets, predicting therapeutic sensitivity of diseased tissues, and identifying early tissue response to pharmacological treatment. These and other recent advances in implementing DESI-MSI as the tool for the development of novel therapies are highlighted in this review.

Serum haptoglobin (Hp), a highly sialylated biomolecule with four N-glycosylation sites, is a positive acute-phase response glycoprotein that acts as an immunomodulator. Hp has gained considerable attention due to its potential as a signature molecule that exhibits aberrant glycosylation in inflammatory disorders and malignancies. Its glycosylation can be analyzed qualitatively and quantitatively by various methods using mass spectrometry. In this review, we have provided a brief overview of Hp structure and biological function and described mass spectrometry-based techniques for analyzing glycosylation ranging from macroheterogeneity to microheterogeneity of Hp in diseases and cancer. The sugars on haptoglobin can be a sweet bridge to link the potential of cancer-specific biomarkers to clinically relevant applications.

Cancers are caused by accumulated DNA mutations. This recognition of the central role of mutations in cancer and recent advances in next-generation sequencing, has initiated the massive screening of clinical samples and the identification of 1000s of cancer-associated gene mutations. However, proteomic analysis of the expressed mutation products lags far behind genomic (transcriptomic) analysis. With comprehensive global proteomics analysis, only a small percentage of single nucleotide variants detected by DNA and RNA sequencing have been observed as single amino acid variants due to current technical limitations. Proteomic analysis of mutations is important with the potential to advance cancer biomarker development and the discovery of new therapeutic targets for more effective disease treatment. Targeted proteomics using selected reaction monitoring (also known as multiple reaction monitoring) and parallel reaction monitoring, has emerged as a powerful tool with significant advantages over global proteomics for analysis of protein mutations in terms of detection sensitivity, quantitation accuracy and overall practicality (e.g., reliable identification and the scale of quantification). Herein we review recent advances in the targeted proteomics technology for enhancing detection sensitivity and multiplexing capability and highlight its broad biomedical applications for analysis of protein mutations in human bodily fluids, tissues, and cell lines. Furthermore, we review recent applications of top-down proteomics for analysis of protein mutations. Unlike the commonly used bottom-up proteomics which requires digestion of proteins into peptides, top-down proteomics directly analyzes intact proteins for more precise characterization of mutation isoforms. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale targeted detection and quantification of important protein mutations are discussed.

Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's disease, inflammation, immune deficiencies, congenital disorders, and cancers. The alterations in the distributions of glycan and glycopeptide isomers are involved in the development and progression of several human diseases. However, the microheterogeneity of glycosylation brings a great challenge to glycomic and glycoproteomic analysis, including the characterization of isomers. Over several decades, different methods and approaches have been developed to facilitate the characterization of glycan and glycopeptide isomers. Mass spectrometry (MS) has been a powerful tool utilized for glycomic and glycoproteomic isomeric analysis due to its high sensitivity and rich structural information using different fragmentation techniques. However, a comprehensive characterization of glycan and glycopeptide isomers remains a challenge when utilizing MS alone. Therefore, various separation methods, including liquid chromatography, capillary electrophoresis, and ion mobility, were developed to resolve glycan and glycopeptide isomers before MS. These separation techniques were coupled to MS for a better identification and quantitation of glycan and glycopeptide isomers. Additionally, bioinformatic tools are essential for the automated processing of glycan and glycopeptide isomeric data to facilitate isomeric studies in biological cohorts. Here in this review, we discuss commonly employed MS-based techniques, separation hyphenated MS methods, and software, facilitating the separation, identification, and quantitation of glycan and glycopeptide isomers.

Top-down proteomics is emerging as a preferred approach to investigate biological systems, with objectives ranging from the detailed assessment of a single protein therapeutic, to the complete characterization of every possible protein including their modifications, which define the human proteoform. Given the controlling influence of protein modifications on their biological function, understanding how gene products manifest or respond to disease is most precisely achieved by characterization at the intact protein level. Top-down mass spectrometry (MS) analysis of proteins entails unique challenges associated with processing whole proteins while maintaining their integrity throughout the processes of extraction, enrichment, purification, and fractionation. Recent advances in each of these critical front-end preparation processes, including minimalistic workflows, have greatly expanded the capacity of MS for top-down proteome analysis. Acknowledging the many contributions in MS technology and sample processing, the present review aims to highlight the diverse strategies that have forged a pathway for top-down proteomics. We comprehensively discuss the evolution of front-end workflows that today facilitate optimal characterization of proteoform-driven biology, including a brief description of the clinical applications that have motivated these impactful contributions.

Recent advances in analytical techniques provide the opportunity to quantify even low-abundance glycopeptides derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Herein, we discuss the sample preparation procedures and the mass spectrometry (MS) strategies that have facilitated glycopeptide quantification, as well as the standards used for glycopeptide quantification. For sample preparation, various glycopeptide enrichment methods are summarized including the columns used for glycopeptide separation in liquid chromatography separation. For MS analysis strategies, MS1 level-based quantification and MS2 level-based quantification are described, either with or without labeling, where we have covered isotope labeling, TMT/iTRAQ labeling, data dependent acquisition, data independent acquisition, multiple reaction monitoring, and parallel reaction monitoring. The strengths and weaknesses of these methods are compared, particularly those associated with the figures of merit that are important for clinical biomarker studies and the pathological and functional studies of glycoproteins in various diseases. Possible future developments for glycopeptide quantification are discussed.

In a world where information-rich methods of analysis are often sought over those with superior figures of merit, there is a constant search for ionization methods which can be applied across diverse analytical systems. The liquid sampling-atmospheric pressure glow discharge (LS-APGD) is a microplasma device which has the inherent capabilities to operate as a combined atomic and molecular (CAM) ionization source. The plasma is sustained by placement of a high voltage (~500 V, dc) onto an electrolytic solution through which the analyte is generally delivered to the discharge. Judicious choice of the solvent provides a means of obtaining atomic/elemental and/or molecular mass spectra. Presented here are the diverse modes of sample introduction and mass spectrometer platforms to which the LS-APGD has been interfaced. Likewise, representative spectra and figures of merit are presented towards elemental and isotope ratio measurements, as well as application to small organic molecules, organometallic complexes, and intact proteins. It is believed that the diversity of analytical applications and ready implementation across the entirety of mass spectrometry platforms portends a level of versatility not realized with other ionization sources.

Pancreatic ductal adenocarcinoma (PDAC) is the most common neoplastic disease of the pancreas, accounting for more than 90% of all pancreatic malignancies. As a highly lethal malignancy, PDAC is the fourth leading cause of cancer-related deaths worldwide with a 5-year overall survival of less than 8%. The efficacy and outcome of PDAC treatment largely depend on the stage of disease at the time of diagnosis. Surgical resection followed by adjuvant chemotherapy remains the only possibly curative therapy, yet 80%-90% of PDAC patients present with nonresectable PDAC stages at the time of clinical presentation. Despite our advancing knowledge of PDAC, the prognosis remains strikingly poor, which is primarily due to the difficulty of diagnosing PDAC at the early stages. Recent advances in glycoproteomics and glycomics based on mass spectrometry have shown that aberrations in protein glycosylation plays a critical role in carcinogenesis, tumor progression, metastasis, chemoresistance, and immuno-response of PDAC and other types of cancers. A growing interest has thus been placed upon protein glycosylation as a potential early detection biomarker for PDAC. We herein take stock of the advancements in the early detection of PDAC that were carried out with mass spectrometry, with special focus on protein glycosylation.

Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly improve the speed and consistency of preparation. When combining robust liquid chromatography-mass spectrometry with either discovery or targeted methods, automated sample preparation facilitates increased throughput and reproducible quantitation of biomarker candidates. These improvements in analysis are also essential to process the large patient cohorts necessary to validate a candidate biomarker for potential clinical use. This article reviews the steps in the workflow, optimization strategies, and known applications in clinical, pharmaceutical, and research fields that demonstrate the broad utility for improved automation of sample preparation in the proteomic field.

This Tutorial is to provide a summary of parameters useful for successful outcomes of laserspray ionization (LSI) and related methods that employ a laser to ablate a matrix:analyte sample to produce highly charged ions. In these methods the purpose of the laser is to transfer matrix-analyte clusters into the gas phase. Ions are hypothesized to be produced by a thermal process where emitted matrix:analyte gas-phase particles/clusters are charged and loss of matrix from the charged particles leads to release of the analyte ions into the gas phase. The thermal energy responsible for the charge-separation process is relatively low and not necessarily supplied by the laser; a heated inlet tube linking atmospheric pressure with the first vacuum stage of a mass spectrometer is sufficient. The inlet becomes the "ion source", and inter alia, pressure, temperature, and the matrix, which can be a solid, liquid, or combinations, become critical parameters. Injecting matrix:analyte into a heated inlet tube using laser ablation, a shockwave, or simply tapping, all produce the similar mass spectra. Applications are provided that showcase new opportunities in the field of mass spectrometry.