Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry

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Resumé

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-of-the-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.
OriginalsprogEngelsk
TidsskriftMolecular and Cellular Proteomics
Vol/bind4
Udgave nummer2
Sider (fra-til)107-19
Antal sider13
ISSN1535-9476
DOI
StatusUdgivet - 1. feb. 2005

Fingeraftryk

Mass spectrometry
Glycoproteins
Gels
Glycosylation
Proteins
Graphite
Powders
Glycopeptides
Electrophoresis
Purification
Polysaccharides
Proteomics

Citer dette

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title = "Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry",
abstract = "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-of-the-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.",
keywords = "Acetonitriles, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Endopeptidase K, Glycopeptides, Glycoproteins, Glycosylation, Graphite, Hexoses, Mass Spectrometry, Ovalbumin, Peptides, Polysaccharides, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Time Factors",
author = "Larsen, {Martin R{\o}ssel} and Peter H{\o}jrup and Peter Roepstorff",
year = "2005",
month = "2",
day = "1",
doi = "10.1074/mcp.M400068-MCP200",
language = "English",
volume = "4",
pages = "107--19",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology",
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TY - JOUR

T1 - Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry

AU - Larsen, Martin Røssel

AU - Højrup, Peter

AU - Roepstorff, Peter

PY - 2005/2/1

Y1 - 2005/2/1

N2 - 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-of-the-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.

AB - 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-of-the-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.

KW - Acetonitriles

KW - Chromatography, High Pressure Liquid

KW - Electrophoresis, Polyacrylamide Gel

KW - Endopeptidase K

KW - Glycopeptides

KW - Glycoproteins

KW - Glycosylation

KW - Graphite

KW - Hexoses

KW - Mass Spectrometry

KW - Ovalbumin

KW - Peptides

KW - Polysaccharides

KW - Proteomics

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Time Factors

U2 - 10.1074/mcp.M400068-MCP200

DO - 10.1074/mcp.M400068-MCP200

M3 - Journal article

C2 - 15561728

VL - 4

SP - 107

EP - 119

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 2

ER -