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Increasing Proteome Coverage Through a Reduction in Analyte Complexity and Its Applications for Single-Cell Proteomics

ABSTRACT: The advancement of sophisticated instrumentation in mass spectrometry has catalyzed a more in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that the oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with a limited quantity of analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion/fractionation are not optimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technology and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a mono-substrate protease for proteomic analysis of samples with low analyte concentrations, an improvement in proteome coverage can be achieved, while maintaining similar quantitative accuracy established by trypsin. This improvement is particularly vital for single-cell samples, where the limited number of protein copies, especially in the context of low-abundance proteins, poses a substantial challenge.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Uterine Cervix

SUBMITTER: Marion Pang

LAB HEAD: Tsui-Fen Chou

PROVIDER: PXD048926 | Pride | 2024-07-17

REPOSITORIES: Pride

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			Action	DRS
	CONS_Marion_20221121_OTE-Aur44_HelaNorm_GluC.csv	Csv
	CONS_Marion_20221121_OTE-Aur44_HelaNorm_lysc.csv	Csv
	CONS_Marion_20221121_OTE-Aur44_HelaNorm_tryp.csv	Csv
	CONS_Marion_20230818_OTE_GluC_AcqTime_Full.csv	Csv
	CONS_Marion_20230818_OTE_LysC_AcqTime_Full.csv	Csv

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Increasing Proteome Coverage Through a Reduction in Analyte Complexity and Its Applications for Single-Cell Proteomics

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