Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation
Year: 2023
Type: article
Abstract: The ubiquity of mass spectrometry-based bottom-up proteomic analyses as a component of biological investigation mandates the validation of methodologies that increase acquisition efficiency, improve sample coverage, and enhance profiling depth. Chromatographic separation is often ignored as an area of potential improvement, with most analyses relying on traditional reversed-phase liquid chromatography (RPLC); this consistent reliance on a single chromatographic paradigm fundamentally limits our view of the observable proteome. Herein, we build upon early reports and validate porous graphitic carbon chromatography (PGC) as a facile means to substantially enhance proteomic coverage without changes to sample preparation, instrument configuration, or acquisition methods. Analysis of offline fractionated cell line digests using both separations revealed an increase in peptide and protein identifications by 43% and 24%, respectively. Increased identifications provided more comprehensive coverage of cellular components and biological processes independent of protein abundance, highlighting the substantial quantity of proteomic information that may go undetected in standard analyses. We further utilize these data to reveal that label-free quantitative analyses using RPLC separations alone may not be reflective of actual protein constituency. Together, these data highlight the value and comprehension offered through PGC-MS proteomic analyses. RAW proteomic data have been uploaded to the MassIVE repository with the primary accession code MSV000091495.
Source: Journal of Proteome Research
Institution: University of Wisconsin–Madison
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funders: National Science Foundation, University of Wisconsin-Madison, National Institutes of Health, National Institute on Aging, NIH Office of the Director, National Institute of Diabetes and Digestive and Kidney Diseases, Division of Graduate Education, National Center for Research Resources, Division of Chemistry, Division of Integrative Organismal Systems
Awards: Acquisition of a High-Field Dual Source FTICR-MS for Pharmaceutical Research, G1803960457, G2075862466, Leveraging Ion Mobility and High Resolution Mass Spectrometry to Probe Neuropeptide Modifications in Environmental Stress, TRTech-PGR: A mass spectrometric-based interdisciplinary approach to deciphering the molecular dialogue between between crop plants and their microbial friends and foes., G555161754, Cellular and molecular mediators of fibrosis in the development of urinary tract dysfunction, G6484839831, G6634682943, Di-Leu-enabled multiplexed quantitation for biomarker discovery and validation in Alzheimer's disease, Acquisition of a High Resolution High Speed MALDI Mass Spectrometer for Biomedical Research at UW-Madison, Acquisition of a Dual-Source, High-Performance, Ion Mobility, Quadrupole Time-of-Flight Mass Spectrometry System for Biomedical Research at UW-Madison, G8699575605, G891322577, G9000041967, Creating a region- specific biomolecular atlas of the brain of Alzheimer’s disease, Mass Spectrometric Studies of Neuropeptides in Feeding, DiLeu-enabled multiplexed quantitation for biomarker discovery and validation in Alzheimer’s disease, G932152812, Probing Protein Structural Changes in Alzheimers Disease
OpenAlex ID: W4387473265