Event Title

Optimization of LC/MS-MS Parameters for Shotgun Proteoanalysis

Presenter Information

Wyatt Schaefer

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Faculty Mentor

Kerfoot Walker, III

Document Type

Poster Presentation

Date of Publication

2021

Abstract

Human aging is increasingly recognized as both a chronological and biological process. Cell senescence, telomere attrition, and lack of proteostasis are just some of the factors that contribute to biological aging2. Additionally, recent literature suggests that the change in concentration of certain proteins in human serum correlates to biological aging3,4. Thus, this research aims to optimize LC/MS2 parameters for shotgun proteoanalysis using a HeLa protein digest standard. We are performing micro-flow LC/MS2-based proteomics. We optimized our LC/MS2 for proteome analysis using a Vanquish Horizon liquid chromatography system directly connected to a Q-Exactive HF-X with BioPharma option mass spectrometer using HeLa Protein Digest Standard. We investigated the effects of spray voltage, probe orientation, column length, protein load, flow rate, gradient time length, and gradient spread on total protein identifications (IDs). Proteins were identified using the UniProtKB Human Reference Proteome database via Proteome Discoverer 2.4 software. A max of 4631 proteins from 24000ng of HeLa digest were identified using a spray voltage of 3.50 kV, neutral/unaltered probe orientation, a gradient length of 180 minutes from 8% acetonitrile to 32% acetonitrile, and a 2.1mm x 150mm column at a 50µl/min flow rate. An optimal protein yield of 3729 was obtained with these parameters with a protein load of 4,000ng. Our data suggests that we are identifying an equal number of proteins with that of the literature1. Thus, we proceed by beginning human serum proteoanalysis. Further research is needed to optimize protein IDs and protein quantitation for human serum proteoanalysis and therefore measure the effects of proteostasis on aging.

Keywords

Human Aging, LC/MS-MS Optimization, Proteoanalysis

Persistent Identifier

http://hdl.handle.net/10950/3061

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Optimization of LC/MS-MS Parameters for Shotgun Proteoanalysis

Human aging is increasingly recognized as both a chronological and biological process. Cell senescence, telomere attrition, and lack of proteostasis are just some of the factors that contribute to biological aging2. Additionally, recent literature suggests that the change in concentration of certain proteins in human serum correlates to biological aging3,4. Thus, this research aims to optimize LC/MS2 parameters for shotgun proteoanalysis using a HeLa protein digest standard. We are performing micro-flow LC/MS2-based proteomics. We optimized our LC/MS2 for proteome analysis using a Vanquish Horizon liquid chromatography system directly connected to a Q-Exactive HF-X with BioPharma option mass spectrometer using HeLa Protein Digest Standard. We investigated the effects of spray voltage, probe orientation, column length, protein load, flow rate, gradient time length, and gradient spread on total protein identifications (IDs). Proteins were identified using the UniProtKB Human Reference Proteome database via Proteome Discoverer 2.4 software. A max of 4631 proteins from 24000ng of HeLa digest were identified using a spray voltage of 3.50 kV, neutral/unaltered probe orientation, a gradient length of 180 minutes from 8% acetonitrile to 32% acetonitrile, and a 2.1mm x 150mm column at a 50µl/min flow rate. An optimal protein yield of 3729 was obtained with these parameters with a protein load of 4,000ng. Our data suggests that we are identifying an equal number of proteins with that of the literature1. Thus, we proceed by beginning human serum proteoanalysis. Further research is needed to optimize protein IDs and protein quantitation for human serum proteoanalysis and therefore measure the effects of proteostasis on aging.