Abstract
A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 5179-5192 |
| Number of pages | 14 |
| Journal | Journal of Proteome Research |
| Volume | 14 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 4 2015 |
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Keywords
- absolute quantitation
- glycopeptide
- immunoglobulin
- site specific glycan analysis
ASJC Scopus subject areas
- Biochemistry
- Chemistry(all)
Cite this
A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins. / Hong, Qiuting; Ruhaak, L. Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual Michael; Lebrilla, Carlito B.
In: Journal of Proteome Research, Vol. 14, No. 12, 04.12.2015, p. 5179-5192.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins
AU - Hong, Qiuting
AU - Ruhaak, L. Renee
AU - Stroble, Carol
AU - Parker, Evan
AU - Huang, Jincui
AU - Maverakis, Emanual Michael
AU - Lebrilla, Carlito B
PY - 2015/12/4
Y1 - 2015/12/4
N2 - A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
AB - A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
KW - absolute quantitation
KW - glycopeptide
KW - immunoglobulin
KW - site specific glycan analysis
UR - http://www.scopus.com/inward/record.url?scp=84949034098&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84949034098&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.5b00756
DO - 10.1021/acs.jproteome.5b00756
M3 - Article
C2 - 26510530
AN - SCOPUS:84949034098
VL - 14
SP - 5179
EP - 5192
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 12
ER -