Hydrazinonicotinic acid derivatization for selective ionization and improved glycan structure characterization by MALDI-MS

Abstract

The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, the signal to noise ratio (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection of sialylated glycan in negative mode, with a 15 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC reagent not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easier due to the omission of a pre-separation step. Importantly, by using different acid reagents as the catalyst, derivatized product signals corresponding to [M + Na](+) or [M + H](+) were obtained respectively, which yield complementary fragmentation patterns for the structure elucidation of glycans. Finally, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.

Recent advances in mass spectrometric analysis of glycoproteins

“…reported direct and effective N‐glycan profiling by simultaneously suppressing the ionization of interfering species, such as peptides, while increasing the ionization efficiency of glycans. This was accomplished by labeling the reducing end of glycans with diazanyl groups using a hydrazinonicotinic acid labeling reagent . The S/N was improved by more than one order of magnitude in both positive‐ and negative‐ion mode.…”

Glycoproteins as diagnostic and prognostic biomarkers for neurodegenerative diseases: A glycoproteomic approach

“…Glycan derivation is often performed to improve detection sensitivity and ionization efficiency by mass spectrometry (Bereman et al., 2010), improve characterization of glycan structures (Jiao et al., 2015), and protect sialic acid residues (Shah et al., 2013). The common methods include reducing end derivation through reductive amination (2‐aminobenzoic acid, 2‐aminobenzamide, et al) or hydrazine chemistry (Gil et al, 2008; Ruhaak et al., 2010), hydroxyl group derivation (Shuang Yang et al., 2013), and sialic acid derivation (Wheeler et al., 2009).…”

Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

“…6‐Hydrazinonicotinic acid (HYNIC) has been introduced as a novel derivatizing reagent for improving the detection sensitivity of glycans (Jiao et al, 2015a). Derivatives were prepared by mixing with either HCl or acetic acid as catalysts.…”