Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies.

The ability to provide an unbiased qualitative and quantitative description of the global changes to proteins in a cell or an organism would permit the systems-wide study of complex biological systems. Label-free quantitative shotgun proteomic strategies (including LC-MS ion intensity quantification and spectral counting) are attractive because of their relatively low cost, ease of implementation, and the lack of multiplexing restrictions when comparing multiple samples. Owing to improvements in the resolution and sensitivity of mass spectrometers, and the availability of analytical software packages, protein quantification by LC-MS ion intensity has increased in popularity. Here, we have addressed the importance of chromatographic alignment on protein quantification, and then assessed how spectral counting compares to ion intensity-based proteomic quantification. Using a spiked-in protein strategy, we analysed two situations that commonly arise in the application of proteomics to cell biology: (i) samples with a small number of proteins of differential abundance in a larger non-changing background, and (ii) samples with a larger number of proteins of differential abundance. To perform these assessments on biologically relevant samples, we used isolated integrin adhesion complexes (IACs). Technical replicate analysis of isolated IACs resulted in a range of alignment scores using the Progenesis QI software package and demonstrated that higher LC-MS chromatographic alignment scores increased the precision of protein quantification. Furthermore, implementation of a simple sample batch-running strategy enabled good chromatographic alignment for hundreds of samples over multiple batches. Finally, we applied the sample batch-running strategy and compared quantification by LC-MS ion intensity to spectral counting and found that quantification by LC-MS ion intensity was more accurate and precise. In summary, these results demonstrate that chromatographic alignment is important for precise and accurate protein quantification based on LC-MS ion intensity and accordingly we present a simple sample re-ordering strategy to facilitate improved alignment. These findings are not only relevant to label-free quantification using Progenesis QI but may be useful to the wide range of MS-based quantification strategies that rely on chromatographic alignment.