Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells).

In-depth LC-MS-based proteomic profiling of limited biological and clinical samples, such as rare cells or tissue sections from laser capture microdissection or microneedle biopsies, has been problematic due, in large, to the inefficiency of sample preparation and attendant sample losses. To address this issue, we developed on-microsolid-phase extraction tip (OmSET)-based sample preparation for limited biological samples. OmSET is simple, efficient, reproducible, and scalable and is a widely accessible method for processing ∼200 to 10,000 cells. The developed method benefits from minimal sample processing volumes (1-3 μL) and conducting all sample processing steps on-membrane within a single microreactor. We first assessed the feasibility of using micro-SPE tips for nanogram-level amounts of tryptic peptides, minimized the number of required sample handling steps, and reduced the hands-on time. We then evaluated the capability of OmSET for quantitative analysis of low numbers of human monocytes. Reliable and reproducible label-free quantitation results were obtained with excellent correlations between protein abundances and the amounts of starting material ( = 0.93) and pairwise correlations between sample processing replicates ( = 0.95) along with the identification of approximately 300, 1800, and 2000 protein groups from injected ∼10, 100, and 500 cell equivalents, resulting from processing approximately 200, 2000, and 10,000 cells, respectively.