While mass spectrometry (MS) is at the core of many glycoproteomic workflows, limitations of the typical data dependent acquisition (DDA) MS experiment mean that often information of lower abundance glycoforms is lost and tools are limited for quantitative analyses. The introduction of sequential windows acquisition of all theoretical fragment ion spectra (SWATH), a data independent acquisition (DIA) MS technique, into the workflow has greatly expanded the scope of quantifiable glycoforms. However due to the technique being relatively young, the toolsets for processing of the huge amount of data produced are still sub-optimal. We would like to introduce two prototype programs targeting limitations in MS/MS programs for glycoproteomic data.
DIALib is a program to be placed at the top of the SWATH data extraction workflow, which aims to complement or replace the usage of the typical DDA based library. DIALib can generate a theoretical ion library currently targeted for PeakView from a simple protein sequence for interrogation of SWATH raw data. Due to its theoretical nature, the DIALib library could potentially cover all glycoforms selected within the chromatographic run of a sample without any information beyond protein/peptide amino acid sequence.
Glypnir is a tool for calculating the relative abundance from AUC values of different N-glycoforms across different complex samples from manually validated data. Glypnir’s input requires output from Byonic software (Protein Metrics) as a node in Proteome Discoverer and user supplied sequence information of the proteins of interest. Using this information, the tool can map any identified precursor back to the protein sequence as well as any accompanying modifications, and group the identified N-glycopeptides by site and monosaccharide composition.
Together DIALib and Glypnir offer an automated solution for different parts of the current glycoproteomic pipelines.