Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry

Abstract

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS 2 (HRMS 2 ) and synchronous precursor selection MS 3 (SPS-MS 3 )-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.

Full-featured, real-time database searching platform enables fast and accurate multiplexed quantitative proteomics

“…When comparing to offline searching using Thermo's high throughput SEQUEST, we observed highly correlated scores similar to previous comparisons of the two search engines (Figure 2A) 10,14 . We tested the speed of Orbiter's RTS using the Hyper two-proteome interference standard 4,6,23 . Searching a full yeast database (6757 protein entries, Uniprot) with a 50ppm precursor tolerance across three isotopes (precursor mass -1/+0/+1) and methionine oxidation as a variable modification and reversed decoy proteins resulted in median search times of 5ms.…”

“…Desired protein amounts were aliquoted and chloroform methanol precipitated, followed by digestion with LysC (overnight at room temperature, vortex speed 2; Wako) and trypsin (6 hours, 37 o C; Promega) digestion. Peptides were labeled with TMT reagents as previously described 6,13 . Labeled peptides were mixed, and dried to remove organic solvent prior to clean-up via Sep-Pak (50mg C18 SepPak; Waters).…”

“…The SPS-MS3 method vastly improved quantitative accuracy, but required the addition of a third quantification scan to every instrument scan cycle which subsequently slowed instrument acquisition speeds 3,4 . While other methods have been developed to reduce precursor co-isolation interference and/or increase the duty cycle speed, these methods generally still rely on either the HRMS2 method or SPS-MS3 method 5,6 . Recently a proof-of-principle showed real-time spectral matching as a novel means to achieve the speed of HRMS2 analyses with the quantitative accuracy of SPS-MS3 4 .…”

Microscaled Proteogenomic Methods for Precision Oncology

“…Analysis time could be reduced with automation of sample processing, the use of faster instrumentation and orthogonal gas phase fraction such as FAIMS [47][48][49] . Furthermore, the protocol as presented can be readily adapted for use as a diagnostic tool by redirecting some of the denatured protein obtained using the BioTExt procedure to PRM assays developed for targets delineated in larger clinical discovery datasets, and, as illustrated for ERBB2 ( Supplementary Figure 6).…”

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

“…This study, which utilized 10 mg of peptide/SILAC state, revealed that lenalidomide causes degradation of the Ikaros transcription factors IKZF1 and IKZF3. To benchmark our new approach, we repeated our previously published SILAC-based experiment by treating MM1S cells with 1 uM lenalidomide for 12h and MG-132 for 3h, or with only MG-132 for 3h using either HCD-MS2, SPS-MS3 22,23 or FAIMS-MS2, a method recently shown to increase the numbers of detected and quantified peptides in proteome studies 24,25 (Figure 3). We then enriched K-ɛ-GG peptides and carried out on-antibody TMT10 labeling for ubiquitylome profiling as described above using just 1 mg of peptide input per sample.…”