Nearly all proteins undergo chemical modifications after translation. These post-translational modifications (PTMs) play crucial roles in functional proteomics, regulating the protein structure, activity, and expression. PTMs regulate interaction with cellular molecules such as nucleic acids, lipids and cofactors, as well as other proteins. PTMs can occur at any moment in the "life cycle" of a protein, influencing their biological function in processes such as initiating catalytic activity, governing protein-protein interactions, or causing protein degradation. Glycosylation and phosphorylation are of particular interest to researchers because they are critical pathways for signaling, activation, and often give insight into disease states.
Analysis of PTMs by mass spectrometry using multiple fragmentation techniques yields the most comprehensive structural characterization of modified proteins. Here we describe useful workflows for analysis of glycosylated and phosphorylated proteins.
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Workflow Overview for Glycosylation
Glycosylation is likely the most common PTM. It is known to play a role in biochemical processes, ranging from mediation of cell interactions to defining cellular identities within complex tissues.1,2 In addition, glycan structures are unique to the proteins and help to regulate the protein activity. Many glycans undergo disease-related expression level changes, potentially providing critical diagnostic information.3,4 Mass spectrometry (MS) has emerged as one of the most powerful tools for structural elucidation of glycosylations due to its sensitive detection and ability to analyze complex mixtures derived from a variety of organisms and cell lines. Sample enrichment, MS acquisition strategy and data analysis must all be optimized to ensure successful glycoproteomics experiments.
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Sample Prep Workflow for Glycosylation
Sample enrichment is central to the success of any MS-based glycoproteomics workflow. It reduces the overall complexity of the sample, thereby facilitating sensitive and accurate analysis. For glycoproteomics, the enrichment steps can be carried out at the protein level1-5, peptide level6-13, or at both levels. It can be targeted or universal, depending on the nature of information sought. For example, targeted enrichment of glycopeptides14,15 or glycoproteins may be performed to selectively isolate a certain subset, on the basis of specific glycan structures. Thermo Scientific Pierce Glycoprotein Isolation Kits, Concanavilin A (ConA) and Wheat Germ Agglutinin (WGA) allow isolation of glycoproteins at the protein level from complex mixtures, including serum, tissue and cultured cell lysates. These complete kits contain the immobilized lectins, binding and wash buffers and columns required to process up to 10 mg of total protein. Thermo Scientific HyperSep™ Retain AX Cartridges enable fast and efficient enrichment of glycopeptides. These cartridges are packed with a high capacity, high purity, highly porous polystyrene DVB material partially modified with quaternary amine functional groups thereby providing excellent efficiency for glycopeptide enrichment.
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Pierce Glycoprotein Isolation Kits
HyperSep™ Retain AX Cartridges
Mass Spectrometry Workflow for Glycosylation
The field of proteomics has benefited tremendously from collisional-activated dissociation (CAD) as this fragmentation technique generates abundant peptide bond cleavages resulting in large number of peptides and protein identifications. However, CAD is not ideal for glycopeptides analysis as this fragmentation does not produce the desired peptide backbone cleavages for sequencing.1 On commercial mass spectrometers CAD fragmentation generates varying degrees of structural information for glycopeptides. Low energy CAD predominantly fragments the glycan on a glycopeptide rather than the peptide, generating spectra that are dominated by glycosidic bond cleavages rather than the desired peptide bond cleavages. Thus, making it very difficult to sequence the glycopeptide.2-5 Further complicating the issue is the cleavage of the peptide-glycan bond, resulting in the loss of information about glycosylation site. The increased collision energy on CAD can generate some peptide backbone fragmentation, but this comes with a complication. It generates mixed MS/MS spectrum where both glycan and peptide information are present making structural interpretation complicated.6 Regardless of whether high- or low-energy CAD is employed, fragmentation of the peptide-glycan bond still occurs limiting the ability to derive information about the site of glycosylation. Electron-transfer dissociation (ETD)7 is far better suited for glycopeptide analyses due to their nonergodic type of dissociation. ETD produces extensive fragmentation of the peptide backbone enabling sequencing of the peptide while preserving glycans on the peptide backbone. This allows for unambiguous assignment of the glycosylation sites, thus providing complementary information to CAD fragmentation. The complementary information provided by ETD along with CAD yields richer glycosylation information than either technique by itself.
Several studies in the past have shown the importance of combining CAD and ETD fragmentation for intact glycopeptides analysis.7-10 However, all of these studies have used both types of fragmentation in a nonselective fashion. We have expanded on this approach to implement an intelligent acquisition strategy termed HCD product-dependent ETD workflow (HCD-pd-ETD) that enables on-the-fly identification of glycopeptides and improves overall productivity of glycopeptide analyses.11-14 In this approach, the mass spectrometer acquires HR/AM HCD spectra in a data-dependent fashion. The instrument identifies glycan oxonium ions on the fly in the HCD spectra and triggers ETD spectra on the glycopeptide precursors only. This results in streamlined data analysis and improvement in dynamic range and duty cycle. The HCD-pd-ETD method is provided within the instrument control software for Orbitrap Fusion and Orbitrap Fusion Lumos mass spectrometers. In addition to HCD-pd-ETD, Orbitrap Fusion and Orbitrap Fusion Lumos MS can trigger any fragmentation based on oxonium ion presence including CID and HCD (HCD-pd-CID, HCD-pd-HCD). Triggering CID fragmentation based on the detection of oxonium ions is useful for elucidating glycan composition information as CID tends to produce more detailed glycan backbone fragmentation. This approach is useful as glycans are heterogeneous PTMs; multiple glycans can be present at a single amino acid site and requires complete characterization of all detected compositions.
Orbitrap Fusion and Orbitrap Fusion Lumos introduced a novel fragmentation referred to as electron-transfer/higher energy collision dissociation (EThcD) that is unique to these platforms. In this fragmentation ETD and HCD are combined in a single spectrum. In EThcD, precursors are fragmented within the linear ion trap using ETD, the precursors, charge reduced precursors and ETD fragment ions are then transferred to the IRM for HCD fragmentation. The result is an EThcD spectrum containing b-, c-, y- and z- ions, a spectrum that is combination of ETD and HCD fragments. Studies have shown that EThcD data provides more complete fragmentation of unmodified and phosphorylated peptides than HCD or ETD alone, and it also increases confidence in localization of phosphorylation sites.15-17 EThcD appears advantageous for glycopeptides, enabling better sequence coverage and glycosylation site localization. It should be noted that EThcD can also be acquired in an HCD-pd- fashion for glycopeptides analysis.
For glycopeptides analysis, LC columns at least 25 cm in length with gradients greater than one hour are recommended.
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Thermo Scientific provides UPLC/HPLC systems that perform at low nano, micro, and high flow rate regimes to meet a wide variety of experimental needs. Thermo Scientific EASY-nLC 1200 and Dionex UltiMate® 3000 RSLCnano LC systems use split-free designs to achieve exceptional stability and reproducibility and they easily couple to all Thermo Scientific mass spectrometers.
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ASMS 2013 Poster
Data Analysis Workflow for Glycosylation
In recent years, much emphasis has been placed on developing bioinformatics tools to simplify interpretation of glycopeptides MSn data. The recent development of a novel software tool, ByonicTM from Protein Metrics, alleviates a lot of the hurdles accompanying manual interpretation. Byonic software utilizes data from both types of fragmentation: HCD data to identify the sugar composition and the corresponding ETD data for peptide backbone information. The final result describes the peptide sequence, site of glycosylation and the glycan composition. Additionally, Byonic also supports the interpretation of EThcD data.
Visit the Protein Metrics website to learn more about Byonic software.
Orbitrap of Choice for Glycosylation
In order to achieve comprehensive structural characterization of glycopeptides, a combination of high-resolution, accurate-mass (HR/AM) mass spectrometry, and both higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) or electron-transfer/higher energy collision dissociation (EThcD) are required. Thermo Scientific Orbitrap Fusion Lumos and Orbitrap Fusion mass spectrometers are capable of these analyses when properly equipped.
The Orbitrap Fusion™ Lumos
- The conventional CID fragmentation available on most commercial mass spectrometers limits structural characterization of glycopeptides. The availability of multiple fragmentation techniques (CID, HCD, ETD and EThcD) on the Orbitrap Fusion Lumos offers a level of versatility required for thorough glycopeptide characterization.
- HCD or CID fragmentation provides glycan composition information while ETD or EThcD is used to both sequence the peptide and determine the site of glycosylation.
- The availability of intelligent acquisition strategies (HCD-pd-any fragmentation) increases the overall productivity of MS analysis for glycopeptides by simplifying analyses and enabling improvements in duty cycle and dynamic range and simplifying data analysis.
- The systems offers the ability to perform “synchronous precursor selection”. Using this approach, multiplexed quantification of glycopeptides in a variety of biological samples is possible and this workflow is only possible on Orbitrap Fusion Lumos OR Orbitrap Fusion mass spectrometers.
Grant Central Resources for Glycosylation
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Comprehensive Characterization of Proteins using a Novel Orbitrap Tribrid Mass Spectrometer — The Game Changer for PTM Analysis
Presentation with Speaker Notes
Thermo Scientific Guide to Glycan Analysis