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Comparative Proteomic Analysis of Capillary and Micropillar Array Columns Using DIA: Insights from the TKO Yeast Standard

ABSTRACT: The effective separation of complex peptide mixtures is a cornerstone of mass spectrometry-based proteomics analysis that enhances the accuracy and depth of proteomic analyses. Here we compare datasets collected of whole cell tryptic peptides which were fractionated by either conventional flame-pulled, C18 packed bed microcapillary columns or microfabricated pillar array columns (μPAC). Sixteen samples from four yeast strains (Δmet6, Δpfk2, Δura2, and wildtype) were analyzed in quadruplicate using data-independent acquisition (DIA). Each column enabled the quantification of >4,700 protein, with >95.4% overlap between column formats. The μPAC showed higher MS1 and MS2 signal intensities, while maintaining similar peptide characteristics as the capillary column. The capillary column favored slightly longer and more hydrophobic peptides. Both columns achieved high data completeness at the protein level (>95%) and reproducible quantification, with μPAC offering slight improvements. Principal component analysis and correlation analysis confirmed the capture of yeast strain-specific differences, with hierarchical clustering prioritizing strain over column effects. Protein quantification validated gene knockouts in both column formats, demonstrating similar accuracy of quantification. These findings highlight the μPAC as a standardized and robust alternative to capillary columns in proteomic analysis.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD063991 | Pride | 2025-11-18

REPOSITORIES: Pride

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			Action	DRS
	Accucore_pepXML.zip	Other
	KEYtoSamples.xlsx	Xlsx
	az06602.raw	Raw
	az06603.raw	Raw
	az06604.raw	Raw

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