Comparative Proteomic Profiling of Human Cell Lines Using Capillary and Micro‐Pillar Array Columns

Chromatographic column selection can impact proteomic profiling, yet comparative studies remain limited. Here, we evaluate the performance of a conventional flame-pulled Accucore packed-bed 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, both 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 cell line-driven patterns, while correlation analyses showed high replicate consistency across column formats. Similarly, 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. SUMMARY: We compare a conventional flame-pulled Accucore resin-packed capillary column and a microfabricated pillar array column (µPAC)-in the context of a TMTpro18-plex experiment using six diverse human cell lines. We evaluate peptide and protein quantification, analytical performance, and reproducibility of both column formats. We demonstrate comparable performance between the two columns and highlight the potential of the robust and standardized µPAC as a viable alternative to traditional capillary columns. These findings offer insights for optimizing column selection in isobaric tag-based proteomic workflows, balancing depth, precision, ease of use, and cost. We provide researchers with evidence-based guidance to enhance experimental design and advance proteomic profiling.