Complex Peptide Mixtures Research Articles

Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF-TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs.

Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.

ITRAQ analysis of hepatic proteins in free-living Mus spretus mice to assess the contamination status of areas surrounding Doñana National Park (SW Spain)

This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas.

(274) Phospholipase A2 of Macrovipera lebetina obtusa venom as a main target to relief pain after snake bites

Snake venoms are complex mixture of proteins and peptides, which comprise about 90-95 % of venom dry weight and other minor components (e.g. ions, carbohydrates, amines etc.). The majority of damages on victims’ organism are caused by viper venoms’ metalloproteinases, serine proteases and phospholipase A2 (svPLA2). Besides heavy damages, snake bites may also cause strong pain sensations. Particularly, pain syndrome is characteristic for the bites of Levantine viper. The main aim of this study was to reveal the mechanism of Macrovipera lebetina obtusa (MLO) venom’s nociceptive action.

Uncovering intense protein diversification in a cone snail venom gland using an integrative venomics approach.

Marine cone snail venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative venomics approach, based on new proteomic results from the venom gland and data previously obtained from the transcriptome and the injectable venom. We describe here the complete peptide content of the dissected venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the venom gland. In addition, by integrating data from three different venom stages, transcriptome, dissected, and injectable venoms, from a single species, we obtain a global overview of the venom processing that occurs from the venom gland tissue to the venom delivery step. In the light of the successive steps in this venom production system, we demonstrate that each venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.

Resolution of protein structure by mass spectrometry.

Typically, mass spectrometry is used to identify the peptides present in a complex peptide mixture and subsequently the precursor proteins. As such, mass spectrometry focuses mainly on the primary structure, the (modified) amino acid sequence of peptides and proteins. In contrast, the three-dimensional structure of a protein is typically determined with protein X-ray crystallography or NMR. Despite the close relationship between these two aspects of protein studies (sequence and structure), mass spectrometry and structure determination are not frequently combined. Nevertheless, this combination of approaches, dubbed conformational proteomics, can offer insight into the function, working mechanism, and conformational status of a protein. In this review, we will discuss the developments at the intersection of mass spectrometry-based proteomics and protein structure determination and start from a brief overview of the classic approaches to identify protein structure along with their advantages and disadvantages. We will subsequently discuss the ability of mass spectrometry to overcome some of the hurdles of these classic methods. Finally, we will provide an outlook on the interplay of mass spectrometry and protein structure determination, and highlight several recent experiments in which mass spectrometry was successfully used to either aid or complement structure elucidation. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:653-665, 2016.

A simple peak detection and label-free quantitation algorithm for chromatography-mass spectrometry.

BackgroundLabel-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.ResultsWe developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.ConclusionsAB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users.

Mass Spectrometry-Based Proteomics for Relative Protein Quantification and Biomarker Identification in Primary Human Hepatocytes.

Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The two-dimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results.

New HPLC Techniques for Proteomics Analysis: A Short Overview of Latest Developments

During the last ten to fifteen years a huge development in the field of analytical techniques used for deciphering the complex nature of the proteome took place. Instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation, and detection become affordable to a broad range of laboratories and new, exciting methods for both separation and detection were published and applied. Newly developed techniques and equipment for separation and detection, such as high pressure nano-HPLC and new techniques for multidimensional HPLC separation, enabled proteomics to experience dynamic growth and enter new paths. Furthermore, proteomics starts entering the “real life” and it is increasingly applied for clinical diagnostics and follow-up of patient's status during the treatment. For any proteomic analysis, one of the most important and sometimes the most difficult task is the separation of the complex mixtures of proteins or peptides prior to their detection and data analysis. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, their benefits and shortcomings which would need far more space than available here.

Using synthetic peptides to benchmark peptide identification software and search parameters for MS/MS data analysis

Tandem mass spectrometry and sequence database searching are widely used in proteomics to identify peptides in complex mixtures. Here we present a benchmark study in which a pool of 20,103 synthetic peptides was measured and the resulting data set was analyzed using around 1800 different software and parameter set combinations. The results indicate a strong relationship between the performance of an analysis workflow and the applied parameter settings. We present and discuss strategies to optimize parameter settings in order to significantly increase the number of correctly assigned fragment ion spectra and to make the analysis method robust.

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.

TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

Nanoparticle-mediated combinatorial targeting of multiple human dendritic cell (DC) subsets leads to enhanced T cell activation via IL-15-dependent DC crosstalk.

Most vaccines depend on coadministration of Ags and adjuvants that activate APCs. Nanoparticles (NPs) have emerged as an attractive vehicle for synchronized delivery of Ags and adjuvants to APCs and can be targeted to specific cell types, such as dendritic cells (DCs), which are potent APCs. Which subset of human DCs should be targeted for optimal activation of T cell immunity, however, remains unknown. In this article, we describe a poly-lactic-coglycolic acid-based NP platform, wherein avidin-decorated NPs can be targeted to multiple human DC subsets via biotinylated Abs. Both BDCA3(+) and monocyte-derived DC-SIGN(+) NP-loaded DCs were equally effective at generating Ag-specific human T cells in culture, including against complex peptide mixtures from viral and tumor Ags across multiple MHC molecules. Ab-mediated targeting of NPs to distinct DC subsets led to enhanced T cell immunity. However, combination targeting to both DC-SIGN and BDCA3(+) DCs led to significantly greater activation of T cells compared with targeting either DC subset alone. Enhanced T cell activation following combination targeting depended on DC-mediated cytokine release and was IL-15 dependent. These data demonstrate that simultaneous targeting of multiple DC subsets may improve NP vaccines by engaging DC crosstalk and provides a novel approach to improving vaccines against pathogens and tumors.

Reprint of “Enhanced Fourier transform for Orbitrap mass spectrometry”

A novel method for processing of periodic signals, which combines absorption spectra presentation with magnitude spectra and finite-impulse-response filtering, is applied to image current transients acquired in Orbitrap mass spectrometry. Phasing of signal for absorption spectra is facilitated by the excitation-by-injection mechanism of forming coherent ion packets in the Orbitrap analyzer. In conjunction with extensive refinement of the trap and electronics design of the Orbitrap analyzer, this method allows completion of the excitation process and initiation of detection within a fraction of a millisecond after ejection of ions from an external storage device into the Orbitrap analyzer, thus avoiding baseline roll and large non-linear phase corrections. For most real-life analytes with limited signal decay over acquisition time, the method achieves a 2-fold increase of resolving power relative to the traditional Fourier transform processing method. For rapidly decaying signals of intact proteins, the relative increase in resolving power is reduced to about 1.4, which accords with theory. Peak shape and mass accuracy in LC/MS measurements obtained employing the novel method appear to be similar to those observed in traditional magnitude-mode FT spectra. Performance of the method was optimized using calibration mixtures and tested on different real-life samples, including complex peptide mixtures and proteins supplied to the mass spectrometer in infusion and LC/MS modes.

Identification of related peptides through the analysis of fragment ion mass shifts.

Mass spectrometry (MS) has become the method of choice to identify and quantify proteins, typically by fragmenting peptides and inferring protein identification by reference to sequence databases. Well-established programs have largely solved the problem of identifying peptides in complex mixtures. However, to prevent the search space from becoming prohibitively large, most search engines need a list of expected modifications. Therefore, unexpected modifications limit both the identification of proteins and peptide-based quantification. We developed mass spectrometry-peak shift analysis (MS-PSA) to rapidly identify related spectra in large data sets without reference to databases or specified modifications. Peptide identifications from established tools, such as MASCOT or SEQUEST, may be propagated onto MS-PSA results. Modification of a peptide alters the mass of the precursor ion and some of the fragmentation ions. MS-PSA identifies characteristic fragmentation masses from MS/MS spectra. Related spectra are identified by pattern matching of unchanged and mass-shifted fragment ions. We illustrate the use of MS-PSA with simple and complex mixtures with both high and low mass accuracy data sets. MS-PSA is not limited to the analysis of peptides but can be used for the identification of related groups of spectra in any set of fragmentation patterns.

Off-Line Multidimensional Liquid Chromatography and Auto Sampling Result in Sample Loss in LC/LC-MS/MS.

Large-scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures. Fractionation can involve ion exchange separation coupled to reversed-phase separation or, more recently, two reversed-phase separations performed at different pH values. When multidimensional separations are combined with tandem mass spectrometry for protein identification, the strategy is often referred to as multidimensional protein identification technology (MudPIT). MudPIT has been used in either an automated (online) or manual (offline) format. In this study, we evaluated the performance of different MudPIT strategies by both label-free and tandem mass tag (TMT) isobaric tagging. Our findings revealed that online MudPIT provided more peptide/protein identifications and higher sequence coverage than offline platforms. When employing an off-line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high-pressure device, a 5.3% loss in protein identifications is observed. When off-line fractionated samples are loaded via an autosampler, a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column. Moreover, peptide recovery was significantly lower after offline fractionation than in online fractionation. Signal-to-noise (S/N) ratio, however, was not significantly altered between experimental groups. It is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces. Therefore, the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples.

Supramolecular disassembly of facially amphiphilic dendrimer assemblies in response to physical, chemical, and biological stimuli.

ConspectusSupramolecular assemblies formed from spontaneous self-assembly of amphiphilic macromolecules are explored as biomimetic architectures and for applications in areas such as sensing, drug delivery, and diagnostics. Macromolecular assemblies are usually preferred, compared with their simpler small molecule counterparts, due to their low critical aggregate concentrations (CAC) and high thermodynamic stability. This Account focuses on the structural and functional aspects of assemblies formed from dendrimers, specifically facially amphiphilic dendrons that form micelle or inverse micelle type supramolecular assemblies depending on the nature of the solvent medium.The micelle type assemblies formed from facially amphiphilic dendrons sequester hydrophobic guest molecules in their interiors. The stability of these assemblies is dependent on the relative compatibility of the hydrophilic and hydrophobic functionalities with water, often referred to as hydrophilic–lipophilic balance (HLB). Disruption of the HLB, using an external stimulus, could lead to disassembly of the aggregates, which can then be utilized to cause an actuation event, such as guest molecule release. Studying these possibilities has led to (i) a robust and general strategy for stimulus-induced disassembly and molecular release and (ii) the introduction of a new approach to protein-responsive supramolecular disassembly. The latter strategy provides a particularly novel avenue for impacting biomedical applications. Most of the stimuli-sensitive supramolecular assemblies have been designed to be responsive to factors such pH, temperature, and redox conditions. The reason for this interest stems from the fact that certain disease microenvironments have aberrations in these factors. However, these variations are the secondary imbalances in biology. Imbalances in protein activity are the primary reasons for most, if not all, human pathology. There have been no robust strategies in stimulus-responsive assemblies that respond to these variations. The facially amphiphilic dendrimers provide a unique opportunity to explore this possibility.Similarly, the propensity of these molecules to form inverse micelles in apolar solvents and thus bind polar guest molecules, combined with the fact that these assemblies do not thermodynamically equilibrate in biphasic mixtures, was used to predictably simplify peptide mixtures. The structure–property relationships developed from these studies have led to a selective and highly sensitive detection of peptides in complex mixtures. Selectivity in peptide extraction was achieved using charge complementarity between the peptides and the hydrophilic components present in inverse micellar interiors. These findings will have implications in areas such as proteomics and biomarker detection.

Enhanced Fourier transform for Orbitrap mass spectrometry

A novel method for processing of periodic signals, which combines absorption spectra presentation with magnitude spectra and finite-impulse-response filtering, is applied to image current transients acquired in Orbitrap mass spectrometry. Phasing of signal for absorption spectra is facilitated by the excitation-by-injection mechanism of forming coherent ion packets in the Orbitrap analyzer. In conjunction with extensive refinement of the trap and electronics design of the Orbitrap analyzer, this method allows completion of the excitation process and initiation of detection within a fraction of a millisecond after ejection of ions from an external storage device into the Orbitrap analyzer, thus avoiding baseline roll and large non-linear phase corrections. For most real-life analytes with limited signal decay over acquisition time, the method achieves a 2-fold increase of resolving power relative to the traditional Fourier transform processing method. For rapidly decaying signals of intact proteins, the relative increase in resolving power is reduced to about 1.4, which accords with theory. Peak shape and mass accuracy in LC/MS measurements obtained employing the novel method appear to be similar to those observed in traditional magnitude-mode FT spectra. Performance of the method was optimized using calibration mixtures and tested on different real-life samples, including complex peptide mixtures and proteins supplied to the mass spectrometer in infusion and LC/MS modes.

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.

Snapshots of scorpion venomics

Scorpions are particularly well adapted to survival in extreme habitats (especially arid and semi-arid environments) and their ability to produce and deliver venoms is an important factor in this success. Scorpion venoms are very complex mixtures of different proteins and peptides. Previous venomics studies revealed that each one of scorpion species may contain more than 100 different peptides. Scorpion venom peptides can be classified into two main types: disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs). The vast majority of DBPs are neurotoxic peptides that specifically interact with various types of ion-channels. The NDBPs have been shown to variously possess bradykinin-potentiating, antimicrobial, hemolytic, cellular signaling and immune-modulating activities. Recently, venom proteomics have been extensively applied in assessing the diversity of scorpion venom from various species. More insights about scorpion venom compositions were also gained through transcriptomic approach. It has provided an opportunity to obtain an overview of the content of scorpion venoms and to compare the relative abundance of toxin transcripts. More importantly, transcriptomics can reflect the biological processes occurring in venom gland cells. This review will highlight recent proteomic and transcriptomic studies to explore the venome of scorpions from different habitats, focusing on desert scorpions from North Africa.

Selective glycopeptide profiling by acetone enrichment and LC/MS

LC/MS is commonly used for site-specific glycosylation analysis of glycoproteins in cells and tissues. A limitation of this technique is the difficulty in acquiring reliable mass spectra for glycopeptides, mainly due to their high heterogeneity and poor hydrophobicity. Here, we establish a versatile method for efficient glycopeptide enrichment to acquire reliable mass spectra. Several lines of evidence using model glycoproteins suggest that our method is based on the different solubility between non-glycosylated and glycosylated peptides in acetone. We also provide data showing that the acetone-precipitated glycopeptide enrichment was successful in acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide in crude human serum. We propose that this method is a powerful tool for the acquisition of reliable mass spectra from trace amounts of glycopeptides and an alternative to lectin affinity enrichment. In this study, we established a versatile method for glycopeptide enrichment to acquire reliable mass spectra because of the limitation of conventional enrichment methods, such as lectin affinity chromatography. Our enrichment method is capable of isolating glycopeptides from complex peptide mixtures such as crude serum.