Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

Benjamin L Parker, Giuseppe Palmisano, Alistair V G Edwards, Melanie Y White, Kasper Engholm-Keller, Albert Lee, Nichollas E Scott, Daniel Kolarich, Brett D Hambly, Nicolle H Packer, Martin R Larsen, Stuart J Cordwell

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.
OriginalsprogEngelsk
TidsskriftMolecular and Cellular Proteomics
Vol/bind10
Udgave nummer8
Sider (fra-til)M110.006833
ISSN1535-9476
DOI
StatusUdgivet - 1. aug. 2011

Fingeraftryk

Myocardial Reperfusion Injury
Glycopeptides
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycosylation
Polysaccharides
Proteins
Liquid chromatography
Liquid Chromatography
Labeling
Glycoproteins
Membrane Proteins
Peptide Hydrolases
Collagen
Microfibrils
Peptides
Laminin
Cadherins
Mast Cells
Integrins
Purification

Citer dette

Parker, B. L., Palmisano, G., Edwards, A. V. G., White, M. Y., Engholm-Keller, K., Lee, A., ... Cordwell, S. J. (2011). Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment. Molecular and Cellular Proteomics, 10(8), M110.006833. https://doi.org/10.1074/mcp.M110.006833
Parker, Benjamin L ; Palmisano, Giuseppe ; Edwards, Alistair V G ; White, Melanie Y ; Engholm-Keller, Kasper ; Lee, Albert ; Scott, Nichollas E ; Kolarich, Daniel ; Hambly, Brett D ; Packer, Nicolle H ; Larsen, Martin R ; Cordwell, Stuart J. / Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment. I: Molecular and Cellular Proteomics. 2011 ; Bind 10, Nr. 8. s. M110.006833.
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abstract = "Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive {"}glycosylations{"} were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.",
author = "Parker, {Benjamin L} and Giuseppe Palmisano and Edwards, {Alistair V G} and White, {Melanie Y} and Kasper Engholm-Keller and Albert Lee and Scott, {Nichollas E} and Daniel Kolarich and Hambly, {Brett D} and Packer, {Nicolle H} and Larsen, {Martin R} and Cordwell, {Stuart J}",
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Parker, BL, Palmisano, G, Edwards, AVG, White, MY, Engholm-Keller, K, Lee, A, Scott, NE, Kolarich, D, Hambly, BD, Packer, NH, Larsen, MR & Cordwell, SJ 2011, 'Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment', Molecular and Cellular Proteomics, bind 10, nr. 8, s. M110.006833. https://doi.org/10.1074/mcp.M110.006833

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment. / Parker, Benjamin L; Palmisano, Giuseppe; Edwards, Alistair V G; White, Melanie Y; Engholm-Keller, Kasper; Lee, Albert; Scott, Nichollas E; Kolarich, Daniel; Hambly, Brett D; Packer, Nicolle H; Larsen, Martin R; Cordwell, Stuart J.

I: Molecular and Cellular Proteomics, Bind 10, Nr. 8, 01.08.2011, s. M110.006833.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

AU - Parker, Benjamin L

AU - Palmisano, Giuseppe

AU - Edwards, Alistair V G

AU - White, Melanie Y

AU - Engholm-Keller, Kasper

AU - Lee, Albert

AU - Scott, Nichollas E

AU - Kolarich, Daniel

AU - Hambly, Brett D

AU - Packer, Nicolle H

AU - Larsen, Martin R

AU - Cordwell, Stuart J

PY - 2011/8/1

Y1 - 2011/8/1

N2 - Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

AB - Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

U2 - 10.1074/mcp.M110.006833

DO - 10.1074/mcp.M110.006833

M3 - Journal article

C2 - 21441315

VL - 10

SP - M110.006833

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 8

ER -