Suppressing the background of LC-ESI-MS analysis of permethylated glycans using the active background ion reduction device

Abstract

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

Keywords: ABIRD; LC–ESI–MS; N‐glycans; background ion reduction; glycomics.

Figure 1.

Fixed scale (A) Chromatographic trace, and (B) Full MS scan of background ions before N-glycan analysis. The black trace represents the ABIRD off condition while the red trace represents the ABIRD on condition.

Figure 2.

(A) Extracted Ion Chromatograms (EICs) of high-mannose N-glycans derived from RNase B in the ABIRD air/off (black trace) and air/on (red trace) condition. The inset bar charts are the quantification comparison (based on intensity, n = 3) of the N-glycans (B) HexNAc2Hex5 (C) HexNAc2Hex6 (D) HexNAc2Hex7 (E) HexNAc2Hex8, and (F) HexNAc2Hex9, in the ABIRD off/on states in combination with the different ESI solvent vapor conditions. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Symbols: , N-acetylglucosamine (GlcNAc); , Galactose (Gal); , Fucose (Fuc); , Mannose (Man); , Glucose (Glc); , N-acetylneuraminic acid (NeuAc/Sialic Acid).

Figure 3.

(A) Extracted Ion Chromatograms (EICs) of sialylated N-glycans derived from HS in the ABIRD MeOH/off (black trace) and MeOH/on (red trace) condition. The inset bar charts are the quantification comparison (based on intensity, n = 3) of the N-glycans (B) HexNAc4Hex5NeuAc1 (C) HexNAc4Hex5NeuAc2 (D) HexNAc5Hex6NeuAc3 (E) HexNAc6Hex7NeuAc4, and (F) HexNAc8Hex6NeuAc1, in the ABIRD off/on states in combination with the different ESI solvent vapor conditions. The asterisks with a bar beneath show the significant change caused by ABIRD. The asterisks without a bar show the significant change between each experimental mode and the ABIRD air/off mode. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Color and symbols as in Figure 2.