Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1

Abstract

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Scheme 1.

Work flow of various methodologies used for O-glycopeptide structural analysis. Numbers in parentheses are laboratory numbers.

Scheme 2.

Work flow of various methodologies used for O-glycan structural analysis. Numbers in parentheses are laboratory numbers. 2-AA, 2-aminobenzoic acid.

Fig. 1.

MALDI profiles of IgA O-glycopeptides of Sap-II, VDS, and NUD samples (lab 1). Spectra were acquired using the linear positive mode. Putative structures were deduced from the molecular mass of glycopeptides and known structures of core 1 O-glycans for the IgA1 hinge region. Average mass was used for these measurements, and mass accuracy was ±1 Da.

Fig. 2.

ESI mass spectra of IgA O-glycopeptides of Sap-II, VDS, and NUD samples (lab 2). a, deconvoluted spectra of three samples. Monoisotopic masses of [M + H]⁺ ions are indicated in the mass spectrum of Sap-II. b, real ESI mass spectrum of Sap-II before deconvolution. The charge state of each peptide ion signal is given in parentheses. Samples were analyzed by LC-MS in positive ion mode using a Paradigm MS4 HPLC system coupled to a Thermo FT-ICR mass spectrometer. Mass accuracy was ±0.1 Da.

Fig. 3.

Comparison of glycan profiles obtained by laboratories having used MS of glycopeptides as analytical tool. Relative abundance of glycan structures was calculated from the signal intensities in the mass spectra of Sap-II (Figs. 1 and 2 and supplemental Figs. S1 and S2). Error bars, 2 S.D. Protonated ions were analyzed. MS mode was MALDI-linear TOF in lab 1 and ESI LTQ-FT-ICR, ESI Orbitrap, and ESI Q-TOF in labs 2, 4, and 5, respectively.