Comparative proteomic profiling of human cell lines using capillary and micro-pillar array column

Chromatographic column selection can impact proteomic profiling, yet comparative studies remain limited. Here, we evaluate the performance of a conventional flame-pulled Accucore resin-packed capillary column and a microfabricated pillar array column (μPAC) in a sample multiplexed global proteome profiling experiment using six human cell lines prepared in triplicate as a TMTpro18-plex. Overall, the chromatography columns exhibited comparable performance. Specifically, the number and overlap of quantified peptides, as well as proteins, was similar between columns. Principal component and hierarchical clustering analyses highlighted reproducible patterns of cell line organization, while correlation analyses showed high replicate consistency across column formats. Similarity, analytical parameters like XCorr scores, signal-to-noise ratio, and peak resolution showed consistency. These findings demonstrate the potential for using robust, standardized microfluidic columns, such as μPAC, in lieu of traditional pull-tipped capillary columns without sacrificing depth or quantitative accuracy. Key advantages of μPAC include its ease of use and durability in a uniform format, although this advantage does come at a higher cost. This comparative analysis offers valuable insights into column selection for TMT-based quantitative proteomics.