Proteomic and phosphoproteomic comparison of human ES and iPS cells.

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis.

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ∼300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.