A Strategy to Achieve Sub-Parts-per-Million Mass Measurement Accuracy of N-Linked Glycans Using Infrared...

Mass calibration techniques are vital in achieving high mass measurement accuracy (MMA) of large biomolecules. Variable ion populations that shift the axial frequencies due to space charge effects have been a significant challenge in achieving sub-parts-per-million (sub-ppm) MMA of glycans on a high-resolution accurate mass (HRAM) orbitrap instrument without the activation of automatic gain control. As the role of glycans is critical to our understanding of diverse biological processes, accurate identification of glycans using sub-ppm MMA is critical for biological interpretations. Hence, this study aims to achieve sub-ppm MMA of glycans by exploring the impact of different ion accumulation times, data collection modes, in addition to custom calibration strategies and external mass correction to optimize accurate mass measurements. Using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), direct analysis was performed on N-linked glycans cleaved from bovine fetuin in negative polarity, where 17 N-linked glycans were detected and annotated. Our results indicate the significance of implementing a custom calibration external lock mass and other techniques, including the effect of external mass correction in achieving sub-ppm MMA of large biomolecules. Implementing such approaches in mass spectrometry imaging (MSI) of biological tissue will enhance the confidence of glycan annotation and enable more accurate biological conclusions.