Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Glycopeptide fragmentation optimization and quantitation by multi collision energy ramp scanning quadrupole data independent acquisition (#71)

Lee A Gethings 1 , Christopher J Hughes 1 , YiJu Chen 2 , YuJu Chen 2 , Robert S Plumb 3 , Johannes PC Vissers 1
  1. Waters Corporation, Wilmslow, CHESHIRE, United Kingdom
  2. Academia Sinica, Taipei, Taiwan
  3. Waters Corporation, Milford, MA, USA

Data-independent acquisition (DIA) approaches have gained increasing popularity due to their high reproducibility. Both targeted and discovery workflows are now routinely used as they can provide both qualitative and quantitative information in a single experiment. Here, we describe and evaluate a novel DIA acquisition method, termed scanning quadrupole DIA, whereby the resolving quadrupole of a high-resolution benchtop Q-TOF instrument is repetitively scanned using overlapping precursor m/z windows to enable both high sensitivity and reproducible acquisition of all fragment ions. LC-MS data were collected in a modified data independent mode of acquisition using continuous quadrupole scanning between m/z 800 to 1600. oa-TOF mass spectra were recorded for each quadrupole position and stored into 200 discrete bins. Three alternating data functions (modes) were acquired: in the low energy MS1 mode, data were collected at 6eV collision energy without quadrupole scanning. In two separate elevated energy MS2 modes, the collision energy was ramped from 12 to 23 eV and from 27 to 50 eV. The spectral acquisition times were between 0.1 s and 0.5 s for each mode. Assessment of the raw data shows that implementation of the hybrid mode of acquisition results in improved fragmentation when compared with data collected using a single collision energy ramp. For each raw data file, in case of untargeted, library-independent searches, two processed peak list files were generated from the individual high-energy CID MS2 data streams. . In-solution digest from tryptic haptoglobulin (HP), a defense response regulator, was used as a model system for method development. Immunoaffinity and HILIC glycopeptide HP enriched plasma sample from three human lung cancer cell lines were contrasted in terms of their relative quantitative glycopeptide profiles. Over 30 potential different glycan structures were characterized, with a significant number showing over expression for cell lines resulting from patients with non-small cell carcinomas.