Glyco-Analytical Multispecific Proteolysis (Glyco-AMP): A Simple Method for Detailed and Quantitative Glycoproteomic Characterization (original) (raw)
Related papers
Analytical Chemistry, 2013
Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N-and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and predetermined protein I.D. (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid deter mination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.
Molecular & Cellular Proteomics, 2013
Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either Nglycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of Nglycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed deN -glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including deN -glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their non-sugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, protein quantification based exclusively upon deglycosylated peptides may lead to inaccurate quantification.
Site-specific protein glycosylation analysis with glycan isomer differentiation
Analytical and Bioanalytical Chemistry, 2011
Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one Nglycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.
Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment
Fundamentals of Glycosylation [Working Title], 2021
High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides.
Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity
International Journal of Molecular Sciences, 2022
Protein glycosylation governs key physiological and pathological processes in human cells. Aberrant glycosylation is thus closely associated with disease progression. Mass spectrometry (MS)-based glycoproteomics has emerged as an indispensable tool for investigating glycosylation changes in biological samples with high sensitivity. Following rapid improvements in methodologies for reliable intact glycopeptide identification, site-specific quantification of glycopeptide macro- and micro-heterogeneity at the proteome scale has become an urgent need for exploring glycosylation regulations. Here, we summarize recent advances in N- and O-linked glycoproteomic quantification strategies and discuss their limitations. We further describe a strategy to propagate MS data for multilayered glycopeptide quantification, enabling a more comprehensive examination of global and site-specific glycosylation changes. Altogether, we show how quantitative glycoproteomics methods explore glycosylation reg...
Molecular & Cellular Proteomics, 2004
Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-ofthe-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.
Glycoproteomics: Past, present and future
FEBS Letters, 2009
This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.
Nature Reviews Methods Primers, 2022
Protein glycosylation refers to the covalent attachment of carbohydrates to polypeptides and represents a class of prevalent and structurally diverse co-translational and post-translational modifications (PTMs) that impact a huge number of biological processes 1-6. Carbohydrate modifications include single monosaccharides and complex carbohydrate chains, both referred to as glycans. Protein glycosylation is a non-templated process and is mediated by enzymes known as glycosyltransferases, responsible for the initiation or elongation of glycans, and oligosaccharyltransferases, responsible for the addition of whole carbohydrate chains. In cells, the complex interplay between glycosyltransferases or oligosaccharyltransferases, carbohydrate transporters and glycosidases-the enzymes that remove these carbohydrates-fine-tunes the glycan structures observed on individual proteins and regulates glycoprotein function, with effects on biological processes that include cellular development 7 , cell-cell communication 8 , hostmicroorganism interactions 9,10 and immunity 5,11,12. For example, the recruitment of leukocytes to sites of inflammation is precisely controlled by specific glycan structures that mediate interactions with cell-surface lectins to enable selective and site-specific leukocyte homing 5,7,11,12. Dysregulation of glycosylation is associated with numerous diseases, including cancer 13-16 , infection and inflammation 17-22 , schizophrenia 23 and a wide range of congenital and neurological disorders 24-26. Unravelling the role of glycosylation under both physiological and pathophysiological conditions is a long-standing goal of glycobiology and has driven the rapid development of methods to track glycosylation for diagnostic and therapeutic purposes 27,28. Glycosylation is a universal protein modification across all domains of life with structurally distinct subclasses and glycan types now recognized 29-34 (Fig. 1a,b). Our knowledge of mammalian asparagine-linked (N-linked) and serine/threonine-linked (O-linked) glycans is the most developed, and these modifications are therefore the focus of this Primer. Characterizing the glycoproteome involves the identification of glycoproteins as well as definition of the macroheterogeneity (structural diversity owing to the presence or absence of glycans at specific glycosylation sites) and microheterogeneity (structural diversity of glycosylation patterns at individual glycosylation sites) 35 within these proteins. Microheterogeneity can arise through differences in the number and type of individual monosaccharide residues within the glycan, the structural arrangements and branching patterns of these monosaccharides Non-templated A process that is not guided by a template, in contrast to templated processes such as DNA transcription and translation.
Overcoming Challenges and Opening New Opportunities in Glycoproteomics
Biomolecules, 2013
Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts ( proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.