Mass spectrometric analysis, automated identification and complete annotation of O-linked glycopeptides - PubMed (original) (raw)

Mass spectrometric analysis, automated identification and complete annotation of O-linked glycopeptides

Zsuzsa Darula et al. Eur J Mass Spectrom (Chichester). 2010.

Abstract

Complex mixtures containing O-linked glycopeptides bearing SA(1-0)GalGalNAc structures, or single GalNAc units were subjected to collision-induced dissociation (CID) and electron transfer dissociation (ETD) analysis on a linear ion trap-Orbitrap mass spectrometer and the resulting data was analyzed using the Protein Prospector software. An overview of the structural information provided by the different fragmentation techniques, as well as their limitations, is presented. We illustrate the importance of the complementary information in the mass spectrometry survey scans as well as the different tandem mass spectrometry techniques. We also present some unique features offered by Protein Prospector that are advantageous in glycopeptide analysis: (i) considering a modification that will produce a neutral loss, without "labeling" the original modification site; (ii) merging CID and ETD search results; (iii) permitting the comparison of different modification site-assignments. Although these data were obtained from secreted glycopeptides, the observations and conclusions are also valid for the intracellular regulatory O-GlcNAc modification.

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Figures

Figure 1

Figure 1

CID and ETD spectra of a 3+ precursor ion at m/z 848.39 (MS spectrum is presented in Figure 4). The upper panel shows the CID spectrum acquired in the linear ion trap; the spectrum is dominated by fragments that consist of the intact glycopeptide minus sugar units, annotated “–SA” (the glycopeptide minus sialic acid), “-SA-Gal”, etc. There are also non-reducing end oxonium ions (“SA”, “SA-Gal”, etc.). The lower panel shows the ETD spectrum. This spectrum contains no useful information, probably because the low charge-density of the precursor ion prevented efficient ETD fragmentation.

Figure 2

Figure 2

ETD spectrum of m/z 733.0369(3+), corresponding to AAT(GalNAcGalSA)LSTLAGQPLLER. This spectrum provides sufficient information for confident sequence identification as well as site assignment. Sialic acid loss from charge-reduced versions of the precursor ion was also detected. Fragments labeled with asterisk indicate hydrogen transfer, i.e. z+1 ions.

Figure 3

Figure 3

This ETD spectrum of m/z 642.03 (4+) was manually deciphered as representing the Na-adduct of TEELQQQNTAPT(GalNAcGalSA)NSPTK. CID and ETD data for the same glycopeptide from a protonated ion of a lower charge state are shown in Figure 1. Its MS spectrum is presented in Figure 4, where the upper panel clearly illustrates that at the higher charge state the solely protonated ion was practically non-existent. Asterisk-labeled fragments retained the Na-ion.

Figure 4

Figure 4

MS survey scan showing the multiply charged precursor ion clusters for the glycopeptide presented in Figures 1 and 3. The MS scan also illustrates that the distribution of protonated ions and Na- and K-adducts can be significantly different at different charge states. Thus, sometimes only metal-ion adducts will be subjected to MS/MS analysis from a cluster.

Figure 5

Figure 5

ETD data from precursor ion 632.6347 (3+), in the linear ion trap. A partially deglycosylated mixture was analyzed, and the ALRPSPT(GalNAc)S(GalNAc)PPSENH glycopeptide was identified from these data. Fragments labeled with asterisk indicate hydrogen transfer, i.e. c-1· and z+1 ions.

Figure 6

Figure 6

CID data of m/z 1157.0476, acquired in the linear ion trap from a partially deglycosylated mixture. Fragment ions labeled with G retained the sugar unit. The structure is DVSASTTVLPDDVT(GalNAc)AYPVG bearing an additional GalNAc unit on any of the 4 other hydroxy amino acids.

Figure 7

Figure 7

Fragment assignment of a glycopeptide CID using MS-Product from SearchCompare output of ProteinProspector. The green y fragment assignments indicate gas-phase deglycosylation, while the glycosylated y5 and y6 ions (in blue) show that there is sufficient information to pinpoint the modification site.

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