: Robust and reproducible large-scale label-free deep quantitative profiling of the MOLM-14 surfaceome using three mass spectrometry platforms over multiple years

Surface proteins are one of the most important and common matrices for clinical chemistry and proteomic analyses. With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free quantitative proteomics has become an increasingly popular tool for profiling global protein abundances. Here, we evaluate the performance of three mass spectrometers, namely Orbitrap Elite (Hybrid Ion Trap-Orbitrap), Q Exactive HF (Hybrid Quadrupole-Orbitrap) and Orbitrap Fusion (Tribrid Quadrupole-Ion Trap-Orbitrap), in label-free semiquantitative analysis of cell surface proteins spanning a four-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With the established workflow, we identify 2335 cell surface proteins in MOLM-14, a human acute myeloid leukemia cell line, with a high degree of reproducibility across three MS platforms over multiple years.