Derivatization by 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry

Abstract

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

“…Glycopeptides from human antithrombin, chicken ovalbumin, and bovine a1-acid glycoprotein could be detected in low fmol amounts (Ullmer, Plematl, & Rizzi, 2006).…”

CE at the omics level: Towards systems biology – An update

“…When a suitable fluorescence labeling method can be linked with LIF detection, sensitivity can be strongly increased by up to 10 215 -10 218 mol for oligosaccharide samples [88]. For intact protein analysis, modified methods using preparative capillary ITP and CZE-ESI-TOF MS have been developed [89][90][91][92], and several kinds of glycoprotein, including ribonuclease B, recombinant human erythropoietin, and ovalbumin have been analyzed [87,93,94]. Intact human erythropoietin glycoforms were analyzed with CZE followed by ESI-TOF MS using capillary coating.…”

Recent Advancement of Chemical Derivatization and Its Applications to High Sensitive Analysis of Peptide in Mass Spectrometry

“…The detection sensitivity of some peptides could be increased over 40-fold via MALDI-TOF MS. Further research indicated that (7): [1123][1124][1125][1126][1127][1128][1129] coumarin derivatization was most pronounced for hydrophilic peptide signal enhancement [5] . Rizzi et al [23] used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to derivatize peptides and evidently enhanced the signal intensity of glycopeptides and small peptides. By the further analysis of enzymatic digestion of a three glycoprotein mixtures (human antithrombin, chicken ovalbumin, and bovine a1-acid-glycoprotein), the limit of detection could be reduced to low fmol amounts.…”