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 (R2 = 0.93) and pairwise
correlations between sample processing replicates (R2 = 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.