Multiple solvent elution, a method to counter the effects of Co-Elution and Ion Suppression of peptides in LC-MS analysis.

ABSTRACT: The abundance of proteins in the cellular proteome ranges from millions of copies to one copy per cell. This wide dynamic range represents a major challenge in proteomic analysis. On average, any human cell type expresses up to approximately 10.000 different protein coding genes that are used to synthesize all the different molecular forms of the protein product from each gene. In a typical shotgun bottom up proteomic approach, all proteins are cleaved enzymatically into peptides which leads to a very complex sample, containing many 100000s of different peptides. These are typically analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a data dependent analysis (DDA). One of the consequences of the high complexity of the samples is that co-elution of peptides cannot be avoided. Due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomics analysis, low abundant peptides are not easily identified [2, 4]. A commonly used strategy to increase the peptide coverage is to reduce the likelihood of co-elution of peptides by using extra-long gradients on nano-LC columns of 50 cm or longer and/or make use of multidimensional separations. In the current study we applied a different method that can noticeably improve the identification of co- eluting peptides. Peptides from a HeLa cell digest were eluted using three different elution solvents (acetonitrile, methanol and acetone) in reverse phase LC-MS/MS shotgun proteomics analysis. Results were compared with three replicates of the same solvent, common practice in shotgun proteomics. In total, we see up to a respectively 10 % and 30% increase in protein and unique peptide combined identifications from the multiple solvent elution when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common over the three runs in the new approach is only 23% respectively compared to 50% in the replicates of the same solvent. The presented method thus provides an easy to implement method to significantly improve the protein coverage in shotgun proteomics without the need to use complex pre fractionation coupled to multidimensional LC set-ups.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Harshavardhan Budamgunta  

LAB HEAD: Geert Baggerman

PROVIDER: PXD011908 | Pride | 2019-06-28

REPOSITORIES: Pride