On the benefits of acquiring peptide fragment ions at high measured mass accuracy

Abstract

The advantages and disadvantages of acquiring tandem mass spectra by collision-induced dissociation (CID) of peptides in linear ion trap Fourier-transform hybrid instruments are described. These instruments offer the possibility to transfer fragment ions from the linear ion trap to the FT-based analyzer for analysis with both high resolution and high mass accuracy. In addition, performing CID during the transfer of ions from the linear ion trap (LTQ) to the FT analyzer is also possible in instruments containing an additional collision cell (i.e., the "C-trap" in the LTQ-Orbitrap), resulting in tandem mass spectra over the full m/z range and not limited by the ejection q value of the LTQ. Our results show that these scan modes have lower duty cycles than tandem mass spectra acquired in the LTQ with nominal mass resolution, and typically result in fewer peptide identifications during data-dependent analysis of complex samples. However, the higher measured mass accuracy and resolution provides more specificity and hence provides a lower false positive ratio for the same number of true positives during database search of peptide tandem mass spectra. In addition, the search for modified and unexpected peptides is greatly facilitated with this data acquisition mode. It is therefore concluded that acquisition of tandem mass spectral data with high measured mass accuracy and resolution is a competitive alternative to "classical" data acquisition strategies, especially in situations of complex searches from large databases, searches for modified peptides, or for peptides resulting from unspecific cleavages.

Figure 1

CID spectra of [M+2H]²⁺ = 632.88 Da, resulting with the identification of peptide GITINTSHVEYDSAVR from Pseudomonas aeruginosa protein PA4265, coded by the gene tufA. A) FT-ICR acquisition. The ion injection time was 319 ms, the total elapsed scan time was 750 ms at resolution 25000. B) Linear ion trap acquisition. The ion injection time was 5 ms, the total elapsed scan time was 230 ms.

Figure 2

CID spectra of [M+2H]²⁺ = 776.87 Da, resulting with the identification of peptide SVLQSWADGEWFK from Pseudomonas aeruginosa protein PA1787, coded by the gene acnB. A) Orbitrap acquisition. The ion injection time was 244 ms, the total elapsed scan time was 490 ms for a resolution of 7500. B) Linear ion trap acquisition. The ion injection time was 14 ms, the total elapsed scan time was 240 ms.

Figure 3

CID spectra of [M+2H]²⁺ = 726.87 Da, resulting with the identification of peptide QIAGAELDAPTPNR from the Pseudomonas aeruginosa protein PA2009, coded by the gene hmgA. A) CID spectrum with Orbitrap acquisition. The ion injection time was 527 ms, the total elapsed scan time was 770 ms at a resolution of 7500. B) C-trap fragmentation spectrum at 50% of the normalized collision energy. The ion injection time was 649 ms, the total elapsed scan time was 890 ms for a resolution of 7500.

Figure 4

ROC-like curves showing sensitivity as a function of 1-specificity of database searches in different conditions and with different CID spectrum acquisition modes. A) Orbitrap acquisitions using different fragment ion m/z tolerance. B) Orbitrap acquisitions versus linear ion trap acquisitions with tryptic specificity and maximum one missed cleavage. C) Orbitrap acquisitions versus linear ion trap acquisitions with half-tryptic specificity and maximum two missed cleavage. D) Orbitrap acquisitions versus linear ion trap acquisitions with half-tryptic specificity, maximum two missed cleavage, and potential modification (methyl-ester) on the amino acids D, E, S and T.

Figure 5

A) Average +/- standard deviation of unique peptide identifications as function of the CID method and data acquisition mode. B) Average +/- standard deviation of identification efficiency (number of identified unique peptides per acquired tandem mass spectrum) as function of the CID method and data acquisition mode.

Figure 6

Distribution of identification quality after database search in open-gap mode (search for unexpected modifications). The search is performed in a database containing all identified Pseudomonas aeruginosa proteins after database search for unmodified peptides, as well as in a random database (containing the double number of randomly retrieved proteins). A) Identified non-sequential sequence tag length distribution with CID and Orbitrap acquisition, searched with 0.01 Da tolerance on fragment ions. B) Identified non-sequential sequence tag length distribution with linear ion trap acquisition, searched with 0.5 Da tolerance on CID fragments. C) Recalculated Phenyx z-score distribution with CID and Orbitrap acquisition, searched with 0.01 Da tolerance on fragment ions. B) Recalculated Phenyx Z-score distribution with linear ion trap acquisition, searched with 0.5 Da tolerance on CID fragments.