GlycoHunter: An Open-Source Software for the Detection and Relative Quantification of INLIGHT-Labeled N-Linked Glycans

Abstract

Glycans are responsible for many biological activities; however, their structures are incredibly diverse and complex, often rendering the field of glycomics unsolvable by a single analytical technique. The development of multiple chemical derivatization strategies and bioinformatic software is responsible for some of the greatest analytical gains in the field of glycomics. The INLIGHT strategy is a chemical derivatization technique using hydrazide chemistry to derivatize the reducing end of Nlinked glycans and incorporates either a natural (NAT, 12 C 6 ) or a stable-isotope label (SIL, 13 C 6 ) to carry out relative quantification. Here we present GlycoHunter, a user-friendly software created in MATLAB that enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pair is generated for relative quantification, including but not limited to, INLIGHT. GlycoHunter accepts the commonly used data file formats imzML and mzXML and effectively identifies all peak pairs associated with NATand SIL-labeled N-linked glycans using MS1 data. It also includes the ability to tailor the search parameters and export the results for further analysis using Skyline or Excel.

Mass Spectrometry Imaging of N-Linked Glycans in a Formalin-Fixed Paraffin-Embedded Human Prostate by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

“…26 GlycoHunter was used to quickly search for multiplycharged peaks detected by IR-MALDESI. 27 Peak pairs with a 0.5017 (z = 2) and 0.3345 Da (z = 3) mass offset were searched in the data with a 10 ppm mass offset m/z tolerance and a minimum abundance threshold of 1000 ions/s. These multiply-charged ions were searched in GlycoMod, a theoretical glycan database, where each identification must be within 2.5 ppm MMA, contain the core N-linked glycan structure (Hex 3 HexNAc 2 ), and not include pentose, KDN, or HexA monosaccharides.…”

“…GlycoHunter was used to quickly search for multiply-charged peaks detected by IR-MALDESI . Peak pairs with a 0.5017 ( z = 2) and 0.3345 Da ( z = 3) mass offset were searched in the data with a 10 ppm mass offset m / z tolerance and a minimum abundance threshold of 1000 ions/s.…”

Imaging of N-Linked Glycans in Biological Tissue Sections Using Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry

“…Next, GlycoHunter was used to create an initial peak list from the experimental nano-DESI MSI data. Singly and doubly charged ions were identified by searching isotopic peaks separated by 1.0 ( z = 1) and 0.5 ( z = 2) m / z in the data with a ±10 ppm m / z tolerance and a minimum abundance threshold of 1000 ions/scan . Candidate assignments were searched with GlycoMod, a theoretical glycan database, where each identification was made with a corresponding match to GlyConnect, an experimentally curated glycomic database.…”

“…Singly and doubly charged ions were identified by searching isotopic peaks separated by 1.0 (z = 1) and 0.5 (z = 2) m/z in the data with a ±10 ppm m/z tolerance and a minimum abundance threshold of 1000 ions/scan. 52 Candidate assignments were searched with GlycoMod, 53 a theoretical glycan database, where each identification was made with a corresponding match to GlyConnect, 54 an experimentally curated glycomic database. The criteria for identification using Glyconnect were as follows: a ±10 ppm m/z tolerance, the presence of Hex 3 HexNAc 2 , and exclusion of pentose, KDN, or HexA monosaccharides.…”

Toolbox Accelerating Glycomics (TAG): Improving Large-Scale Serum Glycomics and Refinement to Identify SALSA-Modified and Rare Glycans

“…Bioinformatics approaches are important in mass spectrometry (MS)-based glycomics, especially database searches [ 10 , 11 ]. Software has been developed by some groups to determine candidate structures of glycans from MS data using tandem MS (MS/MS) [ 12 , 13 , 14 , 15 , 16 ]. While these software packages allow detailed structure determination using MS/MS, they are not suitable for processing a large number of experiments at high speed.…”