18O-Labelling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching
| Type: | Application |
Scientific Paper |
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| Number: | Technology |
L00:3:1100 |
MALDI-TOF |
| Year | Products |
1999 |
Reflex |
| Author | |
Kuster, B., Mann, M.(*) |
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| Reference | |
Anal. Chem. 71(7) 1999 1431-1440 |
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Abstract |
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Glycosylated proteins are in-gel deglycosylated with PNGase F in H 2 18O-contg. medium in order to label partially (50%) the glycosylated Asn residues prior to tryptic digestion and peptide mass mapping by MALDI MS with database searching. Peptides extd. from the gel pieces and present in the in-gel proteolysis supernatant mixt. are also analyzed by nano-electrospray (ESP) MS/MS. For MALDI, a satd. matrix soln. in acetone is mixed 4:1 (v/v) with nitrocellulose in 50% acetone-isopropanol and prepd. by the fast evapn. method. A drop of 10% formic acid is deposited and 0.3-0.5 μl of the digest supernatant is added and allowed to dry. MS1: modified Bruker Reflex, MALDI, TOF, pos. mode, N2 laser, 337 nm. Infusion: desalted peptides in 5% formic acid. MS2: Perkin-Elmer Sciex QqTOF prototype, nano-ESP, pos. mode, CAD: N2, 20-60 eV. Due to isotope labelling, more peptides are detected by MALDI MS to give better database search specificity. Detection of a labelled peptide helps partial sequencing because N-linked carbohydrates are attached solely to Asn residues that form part of the NXS/T sequence. This increases search specificity 100-fold. For all proteins studied (bovine fetuin, human α1-acid glycoprotein, HIVrgp120) glycosylation sites are identified by MALDI MS when 10 pmol glycoprotein is subjected to SDS-PAGE. ESP MS/MS may be used to verify glycosylation site assignment and give detailed sequence information by peptide sequencing of the labelled peptides. An example is given with a peptide from fetuin. |
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