Spectral counting robust on high mass accuracy mass spectrometers

Author(s)

Wolfgang Höhenwarter, Stefanie Wienkoop

Abstract

Mass spectrometry is central to shotgun proteomics, an application that seeks to quantify as much of

the total protein complement of a biological sample as possible. The high mass accuracy, resolution,

capacity and scan rate of modern mass spectrometers have greatly facilitated this endeavor. The sum

of MS to MS/MS transitions in tandem mass spectrometry, the spectral count (SC), of a peptide has

been shown to be a reliable estimate of its relative abundance. However, when using SCs, optimal

MS configurations are crucial in order to maximize the number of low abundant proteins quantified

while keeping the estimates for the highly abundant proteins within the linear dynamic range.

In this study, LC/MS/MS analysis was performed using an LTQ-OrbiTrap on a sample containing

many highly abundant proteins. Tuning the LTQ-OrbiTrap mass spectrometer to minimize redundant

MS/MS acquisition and to maximize resolution of the proteome by accurately measured m/z

ratios resulted in an appreciable increase in quantified low abundant proteins. An exclusion duration

of 90 s and an exclusion width of 10ppm were found best of those tested. The spectral count of

individual proteins was found to be highly reproducible and protein abundance ratios were not

affected by the different settings that were applied. We conclude that on a high mass accuracy

instrument spectral counting is a robust measure of protein abundance even for samples containing

many highly abundant proteins and that tuning dynamic exclusion parameters appreciably improves

the number of proteins that can be reliably quantified.

Organisation(s)

Journal

Rapid Communications in Mass Spectrometry

Volume

Pages

3609-3614

No. of pages

ISSN

0951-4198

Publication date

2010

Peer reviewed

Yes

Austrian Fields of Science 2012

1030 Physics, Astronomy

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