Mass Spectrometry-based Methods Research Articles

Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

Covalent Library Screening by Targeted Mass Spectrometry for Rapid Binding Site Identification.

Interest in covalent drug discovery has surged in recent years, following the high-profile FDA approvals of covalent inhibitors that target BTK and KRAS G12C. High-throughput screening by intact protein mass spectrometry is a popular method for identifying lead matter from covalent fragment libraries. While the technique is proven in its capacity to confirm covalent binding, it does not provide binding site information on its own. Follow-up assays to identify binding sites can be time- and resource-intensive, potentially extending the hit confirmation timeline by weeks or months. Here, we describe the development of CoMPAS, a novel, targeted mass spectrometry-based covalent screening method that provides binding site information in the initial screen. The high sensitivity of targeted detection confers additional advantages over the intact protein method, including the ability to characterize more potent binders and reduced protein reagent requirements. Interpretation of the structure-activity relationship is simplified by enabling the use of binding site-specific EC50 values. To investigate higher-throughput screening beyond what is possible with standard liquid chromatography, we acquired data in parallel on an Agilent RapidFire system and compared the screening results by statistical analysis. To demonstrate the multiplexing capabilities of CoMPAS, we determined the target selectivity of screening hits against a pool of off-target kinases.

Development of Spheroid-FPOP: An In-Cell Protein Footprinting Method for 3D Tumor Spheroids.

Many cancer drugs fail at treating solid epithelial tumors with hypoxia and insufficient drug penetration thought to be contributing factors to the observed chemoresistance. Owing to this, it is imperative to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address this, we developed a mass spectrometry-based protein footprinting method for exploring the impact of hypoxia on protein in 3D colorectal cancer cells. Our group has previously extended the protein footprinting method fast photochemical oxidation of proteins (FPOP) for live cell analysis (IC-FPOP); however, this is the first application of IC-FPOP in a 3D cancer model. In this study, we perform IC-FPOP on intact spheroids (Spheroid-FPOP) using a modified version of the static platform incubator with an XY movable stage (PIXY) FPOP platform. We detected modification in each of three spheroid layers, even the hypoxic core. Pathway analysis revealed protein modifications in over 10 distinct protein pathways, including some involved in protein ubiquitination; a process modulated in cancer pathologies. These results demonstrate the feasibility of Spheroid-FPOP to be utilized as a tool to interrogate protein interactions within a native tumor microenvironment.

Identification of Highly Sensitive Pleural Effusion Protein Biomarkers for Malignant Pleural Mesothelioma by Affinity-Based Quantitative Proteomics.

Malignant pleural mesothelioma (MPM) is an asbestos-associated, highly aggressive cancer characterized by late-stage diagnosis and poor prognosis. Gold standards for diagnosis are pleural biopsy and cytology of pleural effusion (PE), both of which are limited by low sensitivity and markedly inter-observer variations. Therefore, the assessment of PE biomarkers is considered a viable and objective diagnostic tool for MPM diagnosis. We applied a novel affinity-enrichment mass spectrometry-based proteomics method for explorative analysis of pleural effusions from a prospective cohort of 84 patients referred for thoracoscopy due to clinical suspicion of MPM. Protein biomarkers with a high capability to discriminate MPM from non-MPM patients were identified, and a Random Forest algorithm was applied for building classification models. Immunohistology of pleural biopsies confirmed MPM in 40 patients and ruled out MPM in 44 patients. Proteomic analysis of pleural effusions identified panels of proteins with excellent diagnostic properties (90-100% sensitivities, 89-98% specificities, and AUC 0.97-0.99) depending on the specific protein combination. Diagnostic proteins associated with cancer growth included galactin-3 binding protein, testican-2, haptoglobin, Beta ig-h3, and protein AMBP. Moreover, we also confirmed previously reported diagnostic accuracies of the MPM markers fibulin-3 and mesothelin measured by two complementary mass spectrometry-based methods. In conclusion, a novel affinity-enrichment mass spectrometry-based proteomics identified panels of proteins in pleural effusion with extraordinary diagnostic accuracies, which are described here for the first time as biomarkers for MPM.

Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells.

Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.

Perspective on the potential of tandem-ion mobility /mass spectrometry methods for structural proteomics applications.

Cellular processes are usually carried out collectively by the entirety of all proteins present in a biological cell, i.e. the proteome. Mass spectrometry-based methods have proven particularly successful in identifying and quantifying the constituent proteins of proteomes, including different molecular forms of a protein. Nevertheless, protein sequences alone do not reveal the function or dysfunction of the identified proteins. A straightforward way to assign function or dysfunction to proteins is characterization of their structures and dynamics. However, a method capable to characterize detailed structures of proteins and protein complexes in a large-scale, systematic manner within the context of cellular processes does not yet exist. Here, we discuss the potential of tandem-ion mobility / mass spectrometry (tandem-IM/MS) methods to provide such ability. We highlight the capability of these methods using two case studies on the protein systems ubiquitin and avidin using the tandem-TIMS/MS technology developed in our laboratory and discuss these results in the context of other developments in the broader field of tandem-IM/MS.

Validation of a fast and sensitive UPLC-MS/MS quantitative method for N-acyl taurine analysis in biological samples

The recent discovery of N-acyl taurines (NATs) as a class of endogenous bioactive lipids and the perspective of their possible pharmacological applications stimulated the development of mass spectrometry-based methods for their quantitative measurements in biological tissues and fluids. We report here for the first time a procedure validated both in liver surrogate matrix and neat solvent (MeOH) based on UPLC-ESI-QqQ analysis for the identification and quantification of NATs in biological tissue extracts. The LC-MS method was based on five representative lipid analogues, including saturated, monounsaturated and polyunsaturated species, namely N-palmitoyl taurine (C16:0 NAT), N-oleoyl taurine (C18:1 NAT), N-arachidonoyl taurine (C20:4 NAT), N-docosanoyl taurine (C22:0 NAT) and N-nervonoyl taurine (C24:1 NAT), and evaluated for specificity, linearity, matrix effect, recovery, repeatability and intermediate precision and accuracy. The method validated in MeOH by internal standard approach (d4-C20:4 NAT) showed excellent linearity in the range 1–300 ng/ml with R always ≥ 0.9996 for all NATs; intra-day and inter-day precision and accuracy were always within the acceptable range. Specificity was assessed on NAT standards in MeOH, applying the confirmation ratio of two diagnostic MRM ion transitions for product ions at m/z 80 and m/z 107 to true samples in the adopted BEH C18 UPLC conditions. Limit of detection (LOD) and limit of quantification (LOQ) were 0.3–0.4 and 1 ng/ml, respectively, for all compounds. The method was successfully applied to assess the levels of NATs in the mouse liver and, for the first time, in varying sections of the intestine (duodenum, jejunum, ileum and colon). NAT levels increased from duodenum to colon, evidencing a remarkable prevalence in the large intestine of C22:0 NAT, typically occurring mainly in the central nervous system. These findings prompt further studies to disclose the biological function of the various members of this class in different peripheral tissues.

Mass Spectrometry Analysis of DNA and Protein Adducts as Biomarkers in Human Exposure to Cigarette Smoking: Acrolein as an Example.

Acrolein is a major component in cigarette smoke and a product of endogenous lipid peroxidation. It is difficult to distinguish human exposure to acrolein from exogenous sources versus endogenous causes, as components in cigarette smoke can stimulate lipid peroxidation in vivo. Therefore, analysis of acrolein-induced DNA and protein adducts by the highly accurate, sensitive, and specific mass spectrometry-based methods is vital to estimate the degree of damage by this IARC Group 2A carcinogen. This Perspective reviews the analyses of acrolein-induced DNA and protein adducts in humans by mass spectrometry focusing on samples accessible for biomonitoring, including DNA from leukocytes and oral cells and abundant proteins from blood, i.e., hemoglobin and serum albumin.

A Mass Spectrometry-Based Method of Quantifying the Contribution of the Lysine Polymorphism at Position 171 in Sheep PrP.

In sheep, the transmissibility and progression of scrapie, a sheep prion (PrPSc) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrPC). Sheep expressing PrPC with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrPSc. A tryptic peptide, 159R.YPNQVYYRPVDK.Y172, derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-based analysis. This method, using 15N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie-infected sheep. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep.

Molecular insights into sex-specific metabolic alterations in Alzheimer’s mouse brain using multi-omics approach

BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by altered cellular metabolism in the brain. Several of these alterations have been found to be exacerbated in females, known to be disproportionately affected by AD. We aimed to unravel metabolic alterations in AD at the metabolic pathway level and evaluate whether they are sex-specific through integrative metabolomic, lipidomic, and proteomic analysis of mouse brain tissue.MethodsWe analyzed male and female triple-transgenic mouse whole brain tissue by untargeted mass spectrometry-based methods to obtain a molecular signature consisting of polar metabolite, complex lipid, and protein data. These data were analyzed using multi-omics factor analysis. Pathway-level alterations were identified through joint pathway enrichment analysis or by separately evaluating lipid ontology and known proteins related to lipid metabolism.ResultsOur analysis revealed significant AD-associated and in part sex-specific alterations across the molecular signature. Sex-dependent alterations were identified in GABA synthesis, arginine biosynthesis, and in alanine, aspartate, and glutamate metabolism. AD-associated alterations involving lipids were also found in the fatty acid elongation pathway and lysophospholipid metabolism, with a significant sex-specific effect for the latter.ConclusionsThrough multi-omics analysis, we report AD-associated and sex-specific metabolic alterations in the AD brain involving lysophospholipid and amino acid metabolism. These findings contribute to the characterization of the AD phenotype at the molecular level while considering the effect of sex, an overlooked yet determinant metabolic variable.

Monitoring mitochondrial translation by pulse SILAC

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.

A decoupled Virotrap approach to study the interactomes of N-terminal proteoforms.

Given that up to 20% of N-termini of human proteins differ from canonical N-termini as retrieved from sequence databases, a variety of N-terminal proteoforms exists in human cells. These N-terminal proteoforms arise through alternative translation initiation or alternative splicing among others. While such proteoforms diversify the biological functions of the proteome, they remain largely understudied. Recent studies showed that proteoforms expand protein interaction networks by interacting with different prey proteins. As a mass spectrometry-based method to study protein-protein interactions, Virotrap avoids cell lysis by trapping protein complexes in viral-like particles, thereby allowing for the identification of transient and less stable interactions. This chapter describes an adjusted version of Virotrap, decoupled Virotrap, that allows for the detection of interaction partners specific for N-terminal proteoforms.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-β-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS

DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation–histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts.

Mass spectrometry-based techniques for single-cell analysis.

The cell is the most basic structural unit and plays a vital role in the function of an organism. Studying the heterogeneity of cells, especially the qualitative and quantitative analyses of proteins and lipids at the cellular level and even at the subcellular level, is of great significance for the study of some important pathological or physiological processes. Due to the small size of a single cell, low content of analytes and large interference from the biological matrix within the single cell, analytical methods at the single cell level must be highly sensitive and selective. Mass spectrometry is a powerful technology for single-cell analysis, because it has high sensitivity, high selectivity and the ability to monitor multiple chemicals at the same time. In this review, four mass spectrometry-based methods applied to single-cell analysis are introduced and discussed in detail; these are electrospray ionization mass spectrometry (ESI-MS), laser desorption ionization mass spectrometry (LDI-MS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS). The recent advances in single-cell analysis with these mass spectrometry-based techniques are summarized. We believe that this review can provide some help and reference for single-cell analysis by mass spectrometry.

PS-C28-6: HIGH TESTOSTERONE AND LOW ESTRADIOL SERA LEVELS ARE ASSOCIATED WITH UNSTABLE PLAQUE COMPOSITION IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Background and Objectives: Ischemic strokes caused by the rupture of unstable atherosclerotic plaques in the carotid arteries, are leading causes of mortality and disability worldwide. Sex differences exist in plaque composition, where men are more likely to develop unstable plaques than women. Sex hormones affect vascular reactivity and inflammation, but it remains unclear if they are involved in plaque instability and how their role differs in men and women. Herein, we investigated the association between circulating endogenous sex hormone levels and plaque composition/instability in men and women with severe carotid atherosclerosis. Design and method: Pre-operative fasting blood samples and plaque specimens were collected from post-menopausal women and men greater than 55 years old with severe carotid atherosclerosis who underwent a carotid endarterectomy at McGill-affiliated hospitals in Montreal, Canada. Plaque composition and stability were examined by experienced vascular pathologists according to gold-standard histological classifications. Liquid chromatography-tandem mass spectrometry-based methods were developed to quantify endogenous sex hormones, 17ß-estradiol (E2), testosterone, androstenedione, and dehydroepiandrosterone, in sera samples. Results: Subjects (n = 460) had a mean age of 71.0 ± 9.0, were 68.9% male, and had various comorbidities (hypercholesterolemia [82.6%], hypertension [84.6%], type 2 diabetes [34.5%]). Clinical characteristics were similar among post-menopausal women (n = 143) and men (n = 317) with stable versus unstable plaques. Men had a significantly greater proportion of unstable versus stable plaques compared to women (men unstable: 61.5%, women unstable: 41.3%; P < 0.001), characterized predominantly by a large lipid-rich core, hemorrhage, and high presence of inflammatory cell infiltration. Men with unstable plaques had significantly higher levels of testosterone than men with stable plaques (P = 0.004). In men, testosterone levels were inversely associated with fibrosis (P < 0.05), while directly associated with unstable plaque features, including greater lipid core size (P < 0.05), and a greater presence of foam cells (P < 0.05), neovascularization (P < 0.01), and plaque (P < 0.01) and cap inflammation (P < 0.05). No associations were observed between testosterone levels and plaque features in women. Endogenous E2 levels were inversely associated with neovascularization (P < 0.05) and plaque and cap inflammation (P < 0.001) in men, while inversely associated with fibrosis levels in women (P < 0.05). Conclusion: Our findings suggest that higher testosterone and lower E2 levels are associated with unstable plaque composition in men with carotid atherosclerotic disease and may partly explain the higher proportion of unstable plaques observed among men than women of similar age and comorbidity status. Instead, in women, estradiol appears to play a detrimental role in plaque instability by inversely affecting fibrous tissue levels (a key stabilizing feature of plaques).

Discovering Protein-Protein Interactions using Co-Fractionation-Mass Spectrometry with Label-Free Quantitation.

Proteins generally achieve their functions through interactions with other proteins, so being able to determine which proteins interact with which other proteins underlies much of molecular biology. Co-fractionation (CF) is a mass spectrometry-based method for detecting proteome-wide protein-protein interactions. An attractive feature of CF is that it is not necessary to label or otherwise alter samples. Although we have previously published a widely used protocol for a label-incorporated CF methodology, no published protocols currently exist for the label-free variation. In this chapter, we describe a label-free CF-MS protocol. This protocol takes a minimum of a week, excluding the time for cell/tissue culture. It begins with cell/tissue lysis under non-denaturing conditions, after which intact protein complexes are isolated using size exclusion chromatography (SEC) where they are fractionated according to size. The proteins in each fraction are then prepared for mass spectrometry analysis where the constituent proteins are identified and quantified. Finally, we describe an in-house bioinformatics pipeline, PrInCE, to accurately predict protein complexes. Taken together, co-fractionation methodologies combined with mass spectrometry can identify and quantify thousands of protein-protein interactions in biological systems.

Mass spectrometry-based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact-mass level.

The intact-mass MS measurements are becoming increasingly popular in characterization of a range of biopolymers, especially those of interest to biopharmaceutical industry. However, as the complexity of protein therapeutics and other macromolecular medicines increases, the new challenges arise, one of which is the high levels of structural heterogeneity that are frequently exhibited by such products. The very notion of the molecular mass measurement loses its clear and intuitive meaning when applied to an extremely heterogenous system that cannot be characterized by a unique mass, but instead requires that a mass distribution be considered. Furthermore, convoluted mass distributions frequently give rise to unresolved ionic signal in mass spectra, from which little-to-none meaningful information can be extracted using standard approaches that work well for homogeneous systems. However, a range of technological advances made in the last decade, such as the hyphenation of intact-mass MS measurements with front-end separations, better integration of ion mobility in MS workflows, development of an impressive arsenal of gas-phase ion chemistry tools to supplement MS methods, as well as the revival of the charge detection MS and its triumphant entry into the field of bioanalysis already made impressive contributions towards addressing the structural heterogeneity challenge. An overview of these techniques is accompanied by critical analysis of the strengths and weaknesses of different approaches, and a brief overview of their applications to specific classes of biopharmaceutical products, vaccines, and nonbiological complex drugs.

Incorporation of a Disposable ESI Emitter into Inline Cartridge Extraction Mass Spectrometry Improves Throughput and Spectra Stability.

Rapid and reliable methods for detecting tumor margins are crucial for neuro-oncology. Several mass spectrometry-based methods have been recently proposed to address this problem. Inline Cartridge Extraction (ICE) demonstrates the potential for clinical application, based on ex-vivo analysis of dissected tissues, but requires time-consuming steps to avoid cross-contamination. In this work, a method of incorporating a disposable electrospray emitter into the ICE cartridge by PEEK sleeves melting is developed. It reduces total analysis time and improves throughput. The proposed setup also improves the robustness of the ICE molecular profiling as demonstrated with human glial tumor samples in that stability and reproducibility of the spectra were increased.

MALDI-TOF-MS for rapid screening analysis of M-protein in serum.

Monoclonal immunoglobin (M-protein) is a serum biomarker for the diagnosis of plasma cell dyscrasias. Despite limitation of analytical sensitivity and resolution, serum protein electrophoresis and immunofixation electrophoresis are still the front-line tests for the detection of M-proteins. Herein, we developed a MALDI-TOF Mass spectrometry-based method for the screening test of M-proteins in human serum. Based on the unique mass signature of different immunoglobin isotypes, M-Proteins could be rapidly identified and typed. The method demonstrated with high analytical performance and throughput, rapid and simple, which could be a new choice for the diagnosis of plasma cell dyscrasias.