Absolute quantification of human embryonic stem cell transcription factors via stable isotope dilution and selected reaction monitoring mass spectrometry

摘要

Three proteins important to stem cell pluripotency were absolutely quantified in NFF, hES, and iPS cells using selected reaction monitoring. Proteotypic peptides were selected based on both a crude peptide standard screen and recombinant protein trypsin digestions. For Nanog, the triple quadrupole mass spectrometer lacked specifity, therefore the Velos Orbitrap was successfully used as an alternative (OrbiSRM). OrbiSRM was shown to be precise, linear, repeatable, and sensitive. These preliminary results show that this method has higher sensitivity when the analyte is in complex matrix, such as plasma or cell lysate. OrbiSRM holds promise for future quantification work.