Search

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/436434v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@126b92forg.highwire.dtl.DTLVardef@147d6e5org.highwire.dtl.DTLVardef@16d6eb0org.highwire.dtl.DTLVardef@17dfe2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs—all with essentially no missing values across the 16 samples and no loss in quantitative integrity. A set of isobaric labeling reagents called TMTpro enables deep quantitative comparisons of proteome measurements across 16 samples.

Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS1 based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS2 spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides.

Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

In cross-linking mass spectrometry, the depth and sensitivity is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of low abundant cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 23% for a medium complex sample and 56% for strong cation exchange-fractionated HEK293 cell lysate. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot cross-linking data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS

Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs) has become a powerful platform for simultaneous, large-scale phospho-proteome site identification and quantitation. To improve the accuracy of isobaric tag-based quantitation in complex proteomic samples, MS3-based acquisition methods such as Synchronous Precursor Selection (SPS) have been used. However, the method suffers from lower peptide identification rates when applied to enriched phosphopeptide samples compared with unmodified samples due to differences in phosphopeptide fragmentation patterns during tandem MS. We developed and optimized two new acquisition methods for analysis of TMT-labeled multiplexed phosphoproteome samples, which resulted in more phosphopeptide identifications with less ratio distortion when compared with previous methods. We also applied these improved methods to a large-scale study of phosphorylation levels in A549 cell lines treated with insulin or insulin growth factor 1 (IGF-1). Overall, 3378 protein groups and 12 465 phosphopeptides were identified, of which 10 436 were quantified across 10 samples without prefractionation. The accurate measurement enabled us to map to numerous signaling pathways including mechanistic target of rapamycin (mTOR), epidermal growth factor receptor (EGFR, ErbB), and insulin signaling pathways.

Tandem mass tag (TMT)-based quantitation is a strong modality for quantitative proteomics, as samples can be multiplexed, creating large-scale data sets with high precision and minimal missing values. However, coisolation/cofragmentation of near isobaric, coeluting precursor peptide analytes has been well-documented to show ratio compression, compromising the accuracy of peptide/protein quantitation. Advanced peak determination (APD) is a new peak-picking algorithm that shows improved identification of peak detection in survey scans (MS1) to increase the number of precursors selected for unimolecular dissociation (MS2). To increase the number of these "features" selected for MS2 APD purposefully selects multiple peptide precursors of very similar m/ z that often derive from different proteins-a major source of ratio compression in TMT quantification. Here, we evaluate the effects of various data acquisition parameters combined with APD on ratio compression. We find that data acquisition with APD enabled results in more coisolated precursors, more mixed spectra, and in turn, fewer peptide spectral matches, especially at standard on-column loads. We conclude that APD should not be utilized for isobaric tagging, MS2-based experiments.

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). Graphical Abstract ᅟ.

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS2 followed by electron transfer dissociation (ETD)-MS2 upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS2 analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS2 data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the pep...

Top-down mass spectrometry (MS) strategies allow in-depth characterization of proteins by fragmentation of the entire molecule(s) inside a mass spectrometer without requiring prior proteolytic digestion. Importantly, the fragmentation techniques on commercially available mass spectrometers have become more versatile over the past decade, with different characteristics in regards to the type and wealth of fragment ions that can be obtained while preserving labile protein post-translational modifications. Due to these and other improvements, top-down MS has become of broader interest and has started to be applied in more disciplines, such as the quality control of recombinant proteins, analysis and characterization of biopharmaceuticals, and clinical biochemistry to probe protein forms as potential disease biomarkers. This article provides a technical overview and guidance for data acquisition strategies on the Orbitrap platform for single proteins and low complexity protein mixtures. A protein standard mixture composed of six recombinant proteins is also introduced and analysis strategies are discussed in detail. Biological significance The article provides a detailed overview and guidance on how to choose from the variety of available methods for protein characterization by top-down analysis on the Orbitrap platform. Technical details are provided explaining important observations and phenomena when working with intact proteins and data from a number of different samples should serve to provide a solid understanding on how experiments were and should be setup and to set the right expectations on the outcome of these types of experiments. Additionally, a new intact protein standard sample is introduced that will help as a QC sample to check the instrument's hardware and method setup conditions as a requirement for obtaining high quality data from biologically relevant samples.

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

Low-molecular weight heparins (LMWH) prepared by partial depolymerization of unfractionated heparin are used globally to treat coagulation disorders on an outpatient basis. Patent protection for several LMWH has expired and abbreviated new drug applications have been approved by the Food and Drug Administration. As a result, reverse engineering of LMWH for biosimilar LMWH has become an active global endeavor. Traditionally, the molecular weight distributions of LMWH preparations have been determined using size exclusion chromatography (SEC) with optical detection. Recent advances in liquid chromatography–mass spectrometry methods have enabled exact mass measurements of heparin saccharides roughly up to degree-of-polymerization 20, leaving the high molecular weight half of the LMWH preparation unassigned. We demonstrate a new LC–MS system capable of determining the exact masses of complete LMWH preparations, up to dp30. This system employed an ion suppressor cell to desalt the chromatographic effluent online prior to the electrospray mass spectrometry source. We expect this new capability will impact the ability to define LMWH mixtures favorably.

Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pair-wise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and protein-protein interactions at the proteome scale in future studies.

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.

The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. A total of 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

In this study, several glycoproteins: bovine α1-acid glycoprotein, fetuin, and human α1-acid glycoprotein, were analyzed using nanoLC-MS/MS. The performance of three different stationary phases – C8, C18, and porous graphite – was systematically evaluated and optimized for glycopeptide separation. A porous graphite column was chosen as the optimum stationary phase for glycoprotein analysis. An LTQ Orbitrap XL hybrid mass spectrometer equipped with electron transfer dissociation (ETD) was used to acquire, within a single analysis, high-resolution, high-mass-accuracy full MS spectra, collision-induced dissociation (CID) MS/MS spectra for glycan composition analysis, and ETD MS/MS spectra for peptide structure information. As a result, characterization of glycopeptides was achieved within a single LC-MS/MS run.

A MALDI source is interfaced to a modified LTQ Orbitrap XL instrument. This work gives insight into the MALDI source design and shows results obtained with the MALDI source coupled to an accurate mass, high-resolution hybrid mass spectrometer. MALDI-produced ions and fragment ions thereof produced in the mass spectrometer may be analyzed and detected by the Orbitrap analyzer at a maximum mass resolution of 100,000 (FWHM) at m/z 400 with high mass accuracy. An accuracy of ≤2 ppm is achieved by internal mass calibration using lock mass functionality; using external mass calibration, an accuracy of ≤3 ppm is routinely obtained. External mass calibration of the hybrid mass spectrometer is performed using a standard calibration mixture of different peptides and matrix components. The instrumental capabilities are demonstrated for analytical methodologies such as Protein ID using Peptide Mass Fingerprint (PMF) and MS/MS analyses of small molecule samples. Stability of mass accuracy and signal-to-noise ratio for low samples loads (on plates) are demonstrated as well as the experimental dynamic range using α-cyano-4-hydroxy cinnamic acid (CHCA) matrix.