Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of gl...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Analytical chemistry (Washington) 2011-02, Vol.83 (6), p.2029-2037
Hauptverfasser: Wang, Dongdong, Hincapie, Marina, Rejtar, Tomas, Karger, Barry L.
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 2037
container_issue 6
container_start_page 2029
container_title Analytical chemistry (Washington)
container_volume 83
creator Wang, Dongdong
Hincapie, Marina
Rejtar, Tomas
Karger, Barry L.
description Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200–500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ~100 attomole level. The PLOT LC-MS is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.
doi_str_mv 10.1021/ac102825g
format Article
fullrecord <record><control><sourceid>pubmedcentral</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3073582</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>pubmedcentral_primary_oai_pubmedcentral_nih_gov_3073582</sourcerecordid><originalsourceid>FETCH-pubmedcentral_primary_oai_pubmedcentral_nih_gov_30735823</originalsourceid><addsrcrecordid>eNqljb1OwzAUhS0EouVn4A3uCENa21HbTEgoLRQpqEFJ58hNnfQix45sp1J4NkZmnokMCImZ6Qzfd84h5IbRCaOcTUU5RMRn9QkZsxmnwTyK-CkZU0rDgC8oHZEL594oZYyy-TkZcRaGEZ3zMfncKm-Fk9qhx6OE-CCsKL20-C48Gg2mggy9DLJWllhhCU-qL43r1S9-qCrU6HtIOzsYcg9r0XpTK7NDDZU1DSSdriEWupQW0qEotYdUCdcI2DocGKPw9dEATvYTSI01nYNE9IO9aaWGvNt1Sli4TZNNfgdJHCT5axA_L6erfBm8ZFfkrBLKyeufvCT3j6s8Xgdtt2vkvhzurFBFa7ERti-MwOIv0XgoanMsQroIZxEP_z3wDb8UhSQ</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS</title><source>American Chemical Society</source><creator>Wang, Dongdong ; Hincapie, Marina ; Rejtar, Tomas ; Karger, Barry L.</creator><creatorcontrib>Wang, Dongdong ; Hincapie, Marina ; Rejtar, Tomas ; Karger, Barry L.</creatorcontrib><description>Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200–500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ~100 attomole level. The PLOT LC-MS is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/ac102825g</identifier><identifier>PMID: 21338062</identifier><language>eng</language><ispartof>Analytical chemistry (Washington), 2011-02, Vol.83 (6), p.2029-2037</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids></links><search><creatorcontrib>Wang, Dongdong</creatorcontrib><creatorcontrib>Hincapie, Marina</creatorcontrib><creatorcontrib>Rejtar, Tomas</creatorcontrib><creatorcontrib>Karger, Barry L.</creatorcontrib><title>Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS</title><title>Analytical chemistry (Washington)</title><description>Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200–500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ~100 attomole level. The PLOT LC-MS is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.</description><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><recordid>eNqljb1OwzAUhS0EouVn4A3uCENa21HbTEgoLRQpqEFJ58hNnfQix45sp1J4NkZmnokMCImZ6Qzfd84h5IbRCaOcTUU5RMRn9QkZsxmnwTyK-CkZU0rDgC8oHZEL594oZYyy-TkZcRaGEZ3zMfncKm-Fk9qhx6OE-CCsKL20-C48Gg2mggy9DLJWllhhCU-qL43r1S9-qCrU6HtIOzsYcg9r0XpTK7NDDZU1DSSdriEWupQW0qEotYdUCdcI2DocGKPw9dEATvYTSI01nYNE9IO9aaWGvNt1Sli4TZNNfgdJHCT5axA_L6erfBm8ZFfkrBLKyeufvCT3j6s8Xgdtt2vkvhzurFBFa7ERti-MwOIv0XgoanMsQroIZxEP_z3wDb8UhSQ</recordid><startdate>20110221</startdate><enddate>20110221</enddate><creator>Wang, Dongdong</creator><creator>Hincapie, Marina</creator><creator>Rejtar, Tomas</creator><creator>Karger, Barry L.</creator><scope>5PM</scope></search><sort><creationdate>20110221</creationdate><title>Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS</title><author>Wang, Dongdong ; Hincapie, Marina ; Rejtar, Tomas ; Karger, Barry L.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-pubmedcentral_primary_oai_pubmedcentral_nih_gov_30735823</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Dongdong</creatorcontrib><creatorcontrib>Hincapie, Marina</creatorcontrib><creatorcontrib>Rejtar, Tomas</creatorcontrib><creatorcontrib>Karger, Barry L.</creatorcontrib><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Dongdong</au><au>Hincapie, Marina</au><au>Rejtar, Tomas</au><au>Karger, Barry L.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS</atitle><jtitle>Analytical chemistry (Washington)</jtitle><date>2011-02-21</date><risdate>2011</risdate><volume>83</volume><issue>6</issue><spage>2029</spage><epage>2037</epage><pages>2029-2037</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><abstract>Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200–500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ~100 attomole level. The PLOT LC-MS is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.</abstract><pmid>21338062</pmid><doi>10.1021/ac102825g</doi></addata></record>
fulltext fulltext
identifier ISSN: 0003-2700
ispartof Analytical chemistry (Washington), 2011-02, Vol.83 (6), p.2029-2037
issn 0003-2700
1520-6882
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3073582
source American Chemical Society
title Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T06%3A02%3A28IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-pubmedcentral&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Ultrasensitive%20Characterization%20of%20Site-Specific%20Glycosylation%20of%20Affinity%20Purified%20Haptoglobin%20from%20Lung%20Cancer%20Patient%20Plasma%20Using%2010%20%CE%BCm%20i.d.%20Porous%20Layer%20Open%20Tubular%20(PLOT)%20LC-LTQ-CID/ETD-MS&rft.jtitle=Analytical%20chemistry%20(Washington)&rft.au=Wang,%20Dongdong&rft.date=2011-02-21&rft.volume=83&rft.issue=6&rft.spage=2029&rft.epage=2037&rft.pages=2029-2037&rft.issn=0003-2700&rft.eissn=1520-6882&rft_id=info:doi/10.1021/ac102825g&rft_dat=%3Cpubmedcentral%3Epubmedcentral_primary_oai_pubmedcentral_nih_gov_3073582%3C/pubmedcentral%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/21338062&rfr_iscdi=true