A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer

Abstract

Combining source collision-induced dissociation (CID) and tandem mass spectral acquisition in a pseudo-MS 3 experiment using a linear ion trap results in a highly selective and sensitive approach to identifying glycopeptide elution from a protein digest. The increased sensitivity is partially attributed to the nonselective nature of source CID, which allows simultaneous activation of all charge states and coeluting glycoforms generating greater ion abundance for the mass-to-charge (m/z) 204 and/or 366 oxonium ions. Unlike source CID alone, a pseudo-MS 3 approach adds selectivity while improving sensitivity by eliminating chemical noise during the tandem mass spectral acquisition of the oxonium ions in the linear ion trap. Performing the experiments in the hybrid linear ion trap/Fourier transform-ion cyclotron resonance (FT-ICR) enables subsequent high-resolution/high-mass accuracy full-scan mass spectra (MS) and parallel acquisition of MS/MS in the linear ion trap to be completed in 2 s directly following the pseudo-MS 3 scan to collate identification and characterization of glycopeptides in one experimental scan cycle. Analysis of bovine fetuin digest using the combined pseudo-MS 3 , high-resolution MS, and data-dependent MS/MS events resulted in identification of four N-linked and two O-linked glycopeptides without enzymatic cleavage of the sugar moiety or release of the sialic acids before analysis. In addition, over 95% of the total protein sequence was identified in one analytical run. (J Am Soc Mass Spectrom 2006, 17, 168 -179)

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

“…MS signals of these O-linked glycopeptide ions were much lower compared with those of the N-linked glycopeptide ions, largely because of the low glycosylation occupancy (13). When the fetuin tryptic digest was treated with PNGase F to remove the N-linked glycans, the prominent ions detected in the IP-NPLC glycopeptide fraction were all from O-linked glycopeptides of fetuin (r-w) (Fig.…”

“…The tryptic digestion of RNase B yields five N-linked glycopeptides: NLTK-GlcNAc 2 Man 5-9 , where Man is mannose, and GlcNAc is N-acetylglucosamine, with each of the five high mannose type glycans attached to Asn-60 (34). Fetuin has three N-linked sites (Asn-81, Asn1-38, and Asn-158) and four O-linked sites (Ser-253, Thr-262, Ser-264, and Ser-323), which have sialylated glycans attached and low O-glycosylation occupancy (12)(13). Table I shows the non-glycopeptides (i-xxix) sequences of RNase B and fetuin identified from IP-NPLC-MS analysis representing 94% of the amino acid sequence of RNase B and 63% of the amino acid sequence of fetuin.…”

“…1e and Table II) (12,13,23). A typical N-linked carbohydrate derived from a eukaryotic host usually contains 7-25 monosaccharide units (O-linked carbohydrates have a similar size range but with less branching and may be as short as one monosaccharide unit) (4).…”

“…Therefore, the MS 1 signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11)(12)(13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them.…”

Computer simulation of the gap-tripole ion trap with linear injection, 3D ion accumulation, and versatile packet ejection

“…F or analytical sciences, high sensitivity is one of the key points for success. In many types of mass spectrometers, the signal-to-noise ratio increases considerably by analyte accumulation, and the linear ion trap (LIT) concept has been widely used for ion accumulation in hybrid instruments [1][2][3][4][5][6][7]. The LIT has been suitable for the construction of miniature mass analyzers, gas-phase reactions, atomic spectroscopy, precursor fragmentation, and beam physics experiments [8][9][10][11][12][13][14][15][16].…”