Orbitrap: Challenging the Boundaries of Proteomics

In conjunction with the American Society for Mass Spectrometry’s 2013 conference, Thermo Scientific hosted an interactive online seminar to demonstrate the advantages of its novel Orbitrap Fusion Tribrid mass spectrometer.¹ This innovative instrument has the ability to dramatically improve spectral acquisition rates through massive parallelization of the acquisition process, in order to enhance proteome coverage and general experimental throughput.

In his opening remarks, Vlad Zabrouskov described the brief history of proteomics. Notably, researchers have achieved a near-complete proteome coverage in yeast using a shotgun proteomics approach; however, many of the peptide precursors remain inaccessible via traditional tandem mass spectrometry (MS/MS) methods. The Orbitrap Fusion Tribrid mass spectrometer stands to improve future experiments by enhancing sensitivity and throughput: in just two hours, the Orbitrap Fusion Tribrid instrument achieved a near-complete identification of the yeast proteome.

Among the Orbitrap Fusion Tribrid system’s unique features and capabilities are a T-configuration, which allows for a new level of ion manipulation in time and space, and dynamic scan management that permits scan event pipelining and event multitasking, leading to twice the scan rate. In the first full MS scan, ions are isolated in Q1 and then identified in the Orbitrap mass analyzer. The ion routing multipole transfers precursor ions to the ion trap. After these ions are trapped, fragmented and detected in the ion trap, ions from a second ion injection can follow the same analysis pipeline—and so on, with all three analyzers working synchronously.

Additionally, Zabrouskov described an experiment comparing the Orbitrap Fusion Tribrid instrument with the Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer. Using 1 ug of digested HeLa cells in a 140-minute run, when compared to the Orbitrap Elite instrument, the Orbitrap Fusion Tribrid instrument completed five times more MS scans and twice the MS/MS scans in 40 seconds. After the full 140 minutes, the Orbitrap Fusion Tribrid instrument identified 4,996 peptides versus 3,604 peptides identified by the Orbitrap Elite instrument, in half the time.

Post-doctoral researcher Dr. Graeme McAlister described another application of the Orbitrap Fusion Tribrid mass spectrometer, relying on isobaric tandem mass tags (TMTs) in a multiplex quantitation. TMT reagents have led to an increased throughput; however, a cost is associated with them. As Dr. McAlister explained, one issue that arises is a co-isolation of the target protein along with other species, resulting in interference and a distortion of the TMT ratios. One way to alleviate these interferences is to use a MS3 scan to report a purer ion population; however, MS3 alone negatively affects sensitivity.

Using another 8-plex colorectal cancer cell line lysate from cells grown in biological triplicate, Dr. McAlister demonstrated a solution for balancing interference and low sensitivity. The researchers ran the experiment using a MultiNotch MS3 approach, also known as the synchronous precursor scanning feature of the Orbitrap Fusion Tribrid mass spectrometer. The results revealed over 13,000 statistically significant (p<.01) ratios, while the MS2 method measured around 7,000 ratios.

The Orbitrap Fusion Tribrid system has been shown to dramatically improve spectral acquisition rates through massive parallelization of the acquisition process to enhance proteome coverage and general experimental throughput. Based on these results, the Orbitrap Fusion Tribrid mass spectrometer has the potential to challenge the current boundaries of proteomics research.

Reference

1. Zabrouskov, V., and McAlister, G. (2013, October 1) “Pushing the Boundaries of Protein Identification, Characterization, and Quantitation Using a Novel Orbitrap-Based Tribrid MS Architecture,” webinar, spectroscopynow.com.

Post Author: Emily Humphreys. As a biology undergraduate at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.

While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
And has been a regular contributor to Accelerating Science since 2012.