Chen, D. D. Y. | bioRxiv Science

bioRxiv2020

Enrichment-Free Identification of Native Definitive (EnFIND) O-glycoproteome of antibodies in autoimmune diseases

The detection of O-glycosylation at the proteome level has long been a challenging task and a roadblock for O-linked protein glycosylation research. We report an Enrichment-Free Identification of Native Definitive (EnFIND) O-glycoproteome using Trapped Ion Mobility Spectrometry coupled to TOF Mass Spectrometry (TIMS-TOF MS) for direct analysis of protein O-glycosylation in native samples with minimum sample requirement. This approach enabled separation of O-glycopeptide isomers, resolution of O-glycosites and O-glycoform, reduction of sample complexity, and increased sensitivity, thus greatly enhancing analysis of the O-glycoproteome of cell lysates, human serum and exosomes. In addition, we found that antibodies in human serum are highly O-glycosylated on variable, especially hypervariable regions and constant regions, which significantly increases antibody diversity. This method was used to successfully identify characteristic O-glycosylation features of autoimmune diseases.

systems biology↗

bioRxiv2018

RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data from Thermo Orbitrap raw files is demonstrated using the RawTools software. RawTools is a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional preview database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library, and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.

bioinformatics↗