A new algorithm using cross-assignment for label-free quantitation with LC-LTQ-FT MS

Abstract

A new algorithm is described for label-free quantitation of relative protein abundances across multiple complex proteomic samples. Q-MEND is based on the denoising and peak picking algorithm, MEND, previously developed in our laboratory. Q-MEND takes advantage of the high resolution and mass accuracy of the hybrid LTQ-FT MS mass spectrometer (or other high-resolution mass spectrometers, such as a Q-TOF MS). The strategy, termed "cross-assignment", is introduced to increase substantially the number of quantitated proteins. In this approach, all MS/MS identifications for the set of analyzed samples are combined into a master ID list, and then each LC-MS run is searched for the features that can be assigned to a specific identification from that master list. The reliability of quantitation is enhanced by quantitating separately all peptide charge states, along with a scoring procedure to filter out less reliable peptide abundance measurements. The effectiveness of Q-MEND is illustrated in the relative quantitative analysis of Escherichia coli samples spiked with known amounts of non-E. coli protein digests. A mean quantitation accuracy of 7% and mean precision of 15% is demonstrated. Q-MEND can perform relative quantitation of a set of LC-MS data sets without manual intervention and can generate files compatible with the Guidelines for Proteomic Data Publication.

Fig. 1

Flow chart of the Q-MEND algorithm.

Fig. 2. Illustration of the increase in the number of quantitated peptides due to cross-assignment

Quantitation without cross-assignment. Only peptides identified in both samples A and B are used for calculation of relative abundances. Quantitation with cross-assignment. Peptides identified in only one of the samples and visible in the LC/MS of both samples are used for calculation of relative abundances.

Fig. 3. Performance of retention time alignment algorithms

Retention times of common peptides in replicate runs for sample A are compared before and after alignment.

Fig. 4. Run-to-run reproducibility

A. Venn diagram presenting the numbers of PCSs detected and quantitated in replicate runs of sample A (wild type E.coli). B. Reproducibility of PCSs abundances (chromatographic peak areas) in replicate runs of sample A

Fig. 4. Run-to-run reproducibility

A. Venn diagram presenting the numbers of PCSs detected and quantitated in replicate runs of sample A (wild type E.coli). B. Reproducibility of PCSs abundances (chromatographic peak areas) in replicate runs of sample A

Fig. 5. Histogram of coefficient of variation for proteins quantitated with and without cross-assignment

Additional proteins – proteins that were quantitated due to cross-assignment and were not quantitated without cross-assignment.