Complex peptide mixture analysis through ion trap mass spectrometer

1st April 2013

Traditionally, rapid identification of low abundance proteins in complex mixtures has utilised three-dimensional (3D) quadrupole ion trap spectrometry systems.

Recent developments in ion trap mass spectrometry and the introduction of two-dimensional (2D) linear ion trap mass spectrometers provide further tools to boost sample throughput, and produce higher peptide coverage with a higher degree of confidence for protein identification.

<b>High performance mass spectrometry</b>

This study demonstrates how the fast cycle time and unparalleled MS/MS sensitivity of a high performance linear ion trap mass spectrometer result in increased coverage, and faster and more confident protein identification.

LC/MS/MS analysis of enzymatically modified human plasma samples was performed using NanoSpray ionisation. The TurboSEQUEST algorithm was used for data analysis and protein identification.

A whole human plasma sample (5 mg, Sigma) was reduced, alkylated, and enzymatically digested. A total of 3 µL of the digested mixture (1 µg/µL) was loaded onto a reversed-phase (RP) C18 column for LC/MS analysis.

HPLC system: Finnigan Surveyor MS pump from Thermo Electron Corp. with a flow splitter.

Column: 0.15X100 mm C18 (Thermo Electron).

Flow rate: 600 nL/min.

Mobile phase:

A: Water with 0.1per cent Formic Acid

B: Acetonitrile with 0.1 per cent Formic Acid.

Normal gradient: 2 - 65 per cent B in 180 min, 65 - 80 per cent B in 5 min and hold for 5 min, 80 - 2 per cent B in 0.5 min.

Fast gradient: 2 - 65 per cent B in 120 min, 65 - 80 per cent B in 5 min and hold for 5 min, 80 - 2 per cent B in 0.5 min.

Mass Spectrometers: Finnigan LTQ and Finnigan LCQ Deca XP Plus both from Thermo Electron Corp.

Ionisation mode: NanoSpray, positive ion.

Scan sequence: Full-scan MS, Zoom scan, MS/MS scan.

Acquisition modes: Normal, Data Dependent and Dynamic Exclusion.

The zoom scan was used to determine the charge state of each peptide, therefore shortening the time required for data searching analysis. Results were compared to those obtained from the same experiment performed using a conventional 3D ion trap.

<b>Mass spectrometry: Data analysis</b>

Protein identification was performed using the TurboSEQUEST algorithm in the BioWorks 3.1 software package and the Swiss-Prot human database (Swiss Institute of Bioinformatics, Geneva, Switzerland). The identified peptides were further evaluated using charge state versus cross-correlation number (XCorr).

The criteria for positive identification of peptides was XCorr > 1.5 for singly charged ions, XCorr > 2.0 for doubly charged ions, and XCorr > 2.5 for triply charged ions.

The plasma peptide mixture was analysed under two different LC gradients on the Finnigan LTQ and Finnigan LCQ Deca XP Plus.

A four-fold increase in the number of scans was acquired on the Finnigan LTQ in comparison to the Finnigan LCQ Deca XP Plus. This fast scan speed capability, in combination with the unparalleled high sensitivity on the Finnigan LTQ, produces extremely high quality MS/MS spectra, which in turn results in increased confidence of analyte identification.

Moreover, with the rapid duty cycle on the Finnigan LTQ, more experiments and thus more information is obtained from each sample, and in less time. For instance, using the Triple Play feature, with a sequence setup to perform one full-scan, one zoom scan, and one MS/MS scan can be performed in less than 750 milliseconds.

The zoom scan capability is used to determine the charge state of each peptide in order to improve data analysis speed. For example, the zoom scan of the m/z 418 peptide determined that this peptide is doubly charged based on the mass difference of 0.5 Daltons for each sequential peak from the zoom scan.

Another benefit is that high quality MS/MS spectra can be produced from a low abundant ion in the MS spectrum. This added performance provides additional structural information and added confidence in the identification of minor components in a complex mixture.

Increased peptide coverage is also achieved with this higher performance. A two-fold increase in protein coverage was realised for the four proteins analysed.

A dramatic increase (>5 times) in protein identification was also achieved. A direct comparison of results from normal and fast gradients also demonstrates that use of a faster gradient on the LTQ still yields a significantly greater number of protein identifications.

Mass spectrometry: Conclusions

Speed to results and increased confidence in the results are important factors in advancing an investigation or research project.

Using a linear ion trap, increased peptide coverage and protein identification, along with increased confidence in results are achieved in less time.

The rapid scanning and unmatched fast cycle time, in combination with unprecedented high sensitivity of the linear ion trap, yield high quality MS/MS spectra and greater number of scans across an LC peak. This helps to maximise the amount of information obtained from each mass spectrometry analysis.


Melissa Chen, Product Specialist, and Diane Cho, Senior Product Manager, are with Thermo Electron Corporation, Waltham, Massachusetts, USA.




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