Overcoming challenges and opening new opportunities in glycoproteomics

Abstract

Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they are expressed in relatively low abundance in cells. In response, various enrichment methods have been developed to improve the detection of glycoproteins. One such method involves their capture via oxidation of their glycan chains and covalent attachment with hydrazide resins which, when catalyzed by PNGase F, release N-linked glycans and convert the glycosite Asn to Asp; this conversion is identifiable with LC/ESI-MS/MS as a corresponding increase of 0.984 Da in molecular weight. The present study builds on this body of work, providing evidence of three additional strategies that improve glycoprotein identification: (1) use of a high resolution mass spectrometer-the Q Exactive MS-which delivers 2-3 times more glycoprotein identifications than a low resolution MS; (2) optimization of instrument settings and database search parameters to reduce misidentification of N-linked glycopeptides to ~1 percent; and (3) labeling glycopeptides with (18)O during PNGase F treatment to locate N-linked glycosites within peptides containing multiple N-linked sequons.

Keywords: 18O labeling; N-linked glycosites; Orbitrap; deamidation; glycoproteomics; hydrazide-modified magnetic beads; tandem mass spectrometry.

Figure 1

Experimental workflow scheme.

Figure 2

Venn diagrams of the glycoproteins identified by the Q Exactive MS and the linear ion trap-instrument (LTQ) MS from three breast cell lines. All glycoproteins found in LTQ MS analyses were also found in Q Exactive MS analyses.

Figure 3

The LTQ MS CID-MS/MS spectrum (a) and the Q Exactive MS HCD-MS/MS spectrum (b) of the glycopeptide VASVININPN*TTHSTGSCR (AA#248-266, N* denotes a conversion of Asn to Asp for the N-linked site Asn²⁵⁷) from lysosome-associated membrane glycoprotein 2. Both spectra contain a rich set of structural-fragments. The inset figures show the isotopic profile of the doubly charged precursor ion, and the inset tables show lists of the color-coded fragments matching the expected values for various ions. B_n and Y_n denote the N-terminal and the B-terminal fragments, respectively. The MS/MS fragment nomenclature used is according to Johnson et al. [23].

Figure 4

The Q Exactive MS spectrum (a) and the HCD-MS/MS spectrum (b) of the glycopeptide QCN*QTSVCWCVNSVGVR (AA#118-134, N* denotes a conversion of N to D for the N-linked site Asn¹²⁰) from tumor-associated calcium signal transducer 2. The mass difference of 115 Da between y14 and y15 confirms the conversion Asn¹²° to Asp by PNGase F.

Figure 5

The CID-MS/MS spectrum (a) and HCD-MS/MS spectrum (b) of a triply charged precursor ion of the glycopeptide ILLTCSLN*DSATETGHR (AA#153-170, N* denotes a conversion of N to D for the N-linked site Asn¹⁶⁰) from basigin produced by the LTQ MS and the Q Exactive MS, respectively. The CID-MS/MS spectrum (a) contains doubly charged y-series fragments and yields little information for an unambiguous identification of the glycosylation site.

Figure 6

(a) The MS/MS spectrum of the CD166 glycopeptide LNLSEN^#YTLSISNAR (AA#90-144, N^# denotes a conversion of N to D with ¹⁸O for the N-linked site Asn⁹⁵) containing two N-linked consensus sequons. The detected mass difference of 117 Da between y9 and y10 demonstrates that Asn⁹⁵ is deamidated, labeled with ¹⁸O and thus, was glycosylated. (b) The MS/MS spectrum of the zinc-α-2-glycoprotein glycopeptide DIVEYYN^#DSN *GSHVLQGR (AA#103-120, N^# denotes a conversion of N to D with ¹⁸O for the N-linked site Asn¹⁰⁹, and N* denotes a conversion of N to D for the N-linked site Asn¹¹²) that contains two N-linked consensus sequons. The detected mass difference of 117 Da between y11 and y12 demonstrates that the Asn¹⁰⁹ is deamidated and is labeled with ¹⁸O thus is glycosylated. The detected mass difference of 115 Da between y8 and y9 demonstrates that the Asn¹¹² is not glycosylated, and has undergone non-enzymatic deamidation.