Integrating tandem mass tag (TMT)-based multiplexing and chemoproteomics is emerging as a powerful approach for drug discovery. However, sensitivity and throughput remain limiting factors to interrogating full-proteome drug effects. We evaluated the new Orbitrap Ascend mass spectrometer and used it in a multiplexed assessment of whole-proteome drug effects. The Ascend offers new features, including an upstream ion trap for better ion duty cycles and faster FTMSn acquisition rates, and real-time search (RTS) software improvements. Multidimensional comparisons of these features showed significant sensitivity improvement for TMT-based proteomics. With the Ascend we assessed effects of novel inhibitors of peptidyl-prolyl isomerase PIN1 on proteins across the proteome, revealing changes at superior depth.
Biological quadruplicate of four human cell lines were digested with LysC and trypsin, labeled with TMTpro16, pooled, and fractionated on reverse-phase HPLC at high pH. HCT116 cells were treated with PIN1 inhibitors in triplicate at 10 µM for 24 h, then processed in the same manner. A two-proteome mixture containing yeast peptides at defined ratios and human peptides at all 1:1 ratios were prepared in a similar manner without fractionation. Samples were analyzed on the Orbitrap Ascend and the Orbitrap Eclipse using FTMS2 and SPS-MS3 with real-time search. Parameters including sample amount, injection time, sensitivity, and quantitative accuracy and precision were investigated and compared.
Analysis of the two-proteome mixture revealed that quantitative precision was improved with the Orbitrap Ascend. In addition, the Ascend quantified 29% more peptides and 13% more proteins. In both the four-cell-line study and the PIN1 inhibitor study, the Ascend quantified 21–22% more peptides and 6.0–8.1% more proteins (gaining ~500–700 proteins) than the Orbitrap Eclipse while improving quantitative precision. While the Eclipse takes 10 fraction runs to quantify over 8,000 proteins, the Ascend achieves this in only 7 runs. The additional proteins quantified only by the Ascend have lower iBAQ values, suggesting that the Ascend is revealing low-abundant proteins. In the PIN1 inhibitor study, the Ascend identified 175 more statistically significant hits than the Eclipse. One protein not identified by the Eclipse, FGFR1, is involved in some of the same oncogenic pathways as PIN1, revealing potentially relevant compound-target interactions previously not detect. Using the Ascend data, we characterize the families of proteins that are altered by the different PIN1 inhibitors, including on-target and off-target effects.
The improvements within the Orbitrap Ascend enable improved with multiplexed quantitative proteomics and enable deeper characterization of whole-proteome compound effects.