Ion Mobility Tandem Mass Spectrometry Enhances Performance of Bottom-up Proteomics

Abstract

One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A. Molecular & Cellular

A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets

“…The approach scales from small to very large data sets without losing performance, consistently Shotgun proteomics is the most popular approach for large-scale identification and quantification of proteins. The rapid evolution of high-end mass spectrometers in recent years (1)(2)(3)(4)(5) has made proteomic studies feasible that identify and quantify as many as 10,000 proteins in a sample (6 -8) and enables many lines of new scientific research including, for example, the analysis of many human proteomes, and proteome-wide protein-drug interaction studies (9 -11). One fundamental step in most proteomic experiments is the identification of proteins in the biological system under investigation.…”

Extracting histones for the specific purpose of label‐free MS

“…Subsequently, peptides were transferred to an analytical column (100 μm × 100 mm nanoACQUITY UPLC 1.7 μm Peptide BEH, Waters) and separated at a flow rate of 300 nL/min using a gradient of 60 min going from 1 to 40% buffer B (0.1% formic acid in acetonitrile). MS data acquisition parameters were set according to Helm et al., with minor adaptations . A Q‐TOF SYNAPT G2‐S i instrument (Waters) was operated in positive mode for High Definition‐DDA, using a nano‐ESI source, acquiring full scan MS and MS/MS spectra ( m/z 50–5000) in resolution mode.…”

Proteome sequencing goes deep

“…Using current technology, 9,500 proteins can be identified from just 100 nL of formalin-fixed and paraffin-embedded (FFPE) tissue [28•]. Although it is impossible to ionize and sequence every peptide, efficient sample preparation, coupled with advances in MS instrumentation [29–31], separation methodology [32–35], and fragmentation techniques [36–39] have vastly increased the observable portion of the human proteome.…”

“…Ion mobility coupled with MS has also been explored as an option for decreasing sample complexity and improving identification efficiency [42]. Traveling wave ion mobility spectrometry (TWIMS) significantly improved the duty-cycle of a time-of-flight (TOF) instrument, identifying ~7,500 protein groups from HeLa cells in one day of analysis time [31]. …”