MSACL 2023 Abstract

High flow LC-MS analysis of plasma digest for protein identification Richard Gibson, Stephanie Samra, Yvonne Song, Philip Remes, Cristina Jacob Thermo Scientific, San Jose, CA Richard Gibson, PhD (Presenter) Thermo Fisher Scientific

Presenter Bio: Richard Gibson is an applications scientist for Thermo Fisher Scientific, based out of San Jose, California. Richard studied mathematics at the University of York and the University of St Andrews before transitioning to biochemistry. He completed his PhD on the biochemical and biophysical characterization of the quartromicin polyketide synthase at the University of Warwick and now works on a range of applications, including peptide quantitation by LC-MS.

Introduction: Human proteome analysis through liquid chromatography mass spectrometry (LC-MS) can play an important role in biomarker discovery in matrices such as plasma, serum or urine. Analyzing digested human plasma allows the detection of 1,000s of peptides within one run. Bioinformatic analysis then allows the identification of signature peptides that may be used to identify protein biomarkers of a disease. Data from high flow rate LC-MS has been shown to be able to identify 100s of proteins within one run that may then be used as targets for future studies.
MS2 scans can be data-dependent (DDA), data-independent (DIA) or targeted, such as with parallel reaction monitoring (PRM). Each of these scan types is accessible through high resolution accurate mass (HRAM) mass spectrometry (MS) and have been demonstrated to efficiently screen for proteins in plasma.

Methods: Plasma samples (heparin tubes) from a single donor obtained with IRB approval were centrifuged (800 RCF, 10 minutes). Plasma was mixed and transferred to 1.5 mL heparin tubes in 1 mL aliquots. An AB-1300 200 uL PCR plate is filled with 100 uL per well of 8 M GuHCl, 275 mM Tris-Hcl, 2% n-propanol, 10 mM DTT, pH 8.6. Plasma samples were vortexed and 30uL was added to each well. Samples were then mixed by pipetting. The plate was sealed using the Easypeel foil (AB-3739) on an APLS 3000 plate sealer and heated (37 oC, 1 hour). The seal was then removed, and 4.5 uL of 1M iodoacetic acid sodium salt (acros organics) was added to each well. The plate was then resealed, vortexed (1200 rpm, 5 minutes) and centrifuged (2500 RCF, 2 minutes). Nunc 2.2 mL deepwell plates (Nunc, 278752) were filled with 1.7 mL of 50 mM Tris-Hcl, 5 mM CaCl2. Samples were transferred to the deepwell plates containing 120uL of Pierce TPCK trypsin, 1mg/mL in 25 mM acetic acid in each well. The plates resealed and incubated (37 oC, 5 hours), after which 100 uL of acetic acid was added to each well, and the plates were resealed, vortexed and centrifuged (2500 RCF, 20 minutes).

From each sample 45 ug of digested plasma was injected into a VanquishTM Horizon UHPLC system fitted with a 2.1 x 50 mm PS-DVB trap column, and an AcclaimTM C18 120 Bonded phase 2.1 x 250 mm C18 2.2 um 120A pore analytical column with a 52 minute gradient and a flow rate of 250 uL/minute. The Orbitrap ExplorisTM 240 was set to a 50% AGC target value for the full scan with EASYIC. DDA, DIA and PRM data were collected in triplicate from various optimized MS2 scans. PRM precursor transitions were generated from a library created from DIA gas phase fractionation data.

Data was processed with Thermo Scientific Proteome DiscovererTM 3.0 and Skyline software.

Results: Protein and peptide IDs were determined from DDA, DIA and PRM scans. Precursor transitions for the PRM scans were chosen using seven small window gas phase fractionation (GPF) DIA scans. Selectively reducing the database size was demonstrated to increase the resolving power of the identification method, significantly increasing the number of protein and peptide IDs for DDA and PRM scans.

Conclusions: Protein and peptide IDs were calculated for DDA, DIA and PRM data, highlighting the versatility of HRAM mass spectrometry. Successful collection of DDA, DIA and PRM data highlights orbitrap technology being suitable for acquiring discovery data and translating it to a targeted method on the same instrument.