Simple and Integrated Spintip-Based Technology Applied for Deep Proteome Profiling

Great
efforts have been taken for developing high-sensitive mass
spectrometry (MS)-based proteomic technologies, among which sample
preparation is one of the major focus. Here, a simple and integrated
spintip-based proteomics technology (SISPROT) consisting of strong
cation exchange beads and C18 disk in one pipet tip was
developed. Both proteomics sample preparation steps, including protein
preconcentration, reduction, alkylation, and digestion, and reversed
phase (RP)-based desalting and high-pH RP-based peptide fractionation
can be achieved in a fully integrated manner for the first time. This
easy-to-use technology achieved high sensitivity with negligible sample
loss. Proteomic analysis of 2000 HEK 293 cells readily identified
1270 proteins within 1.4 h of MS time, while 7826 proteins were identified
when 100000 cells were processed and analyzed within only 22 h of
MS time. More importantly, the SISPROT can be easily multiplexed on
a standard centrifuge with good reproducibility (Pearson correlation
coefficient > 0.98) for both single-shot analysis and deep proteome
profiling with five-step high-pH RP fractionation. The SISPROT was
exemplified by the triplicate analysis of 100000 stem cells from human
exfoliated deciduous teeth (SHED). This led to the identification
of 9078 proteins containing 3771 annotated membrane proteins, which
was the largest proteome data set for dental stem cells reported to
date. We expect that the SISPROT will be well suited for deep proteome
profiling for fewer than 100000 cells and applied for translational
studies where multiplexed technology with good label-free quantification
precision is required.