Comparative evaluation of small molecular additives and their effects on peptide/protein identification

Detergents and salts are widely used in lysis buffer to enhance protein extraction from biological samples, facilitating in-depth proteome analysis, however, to efficiently remove these detergent and salt additives from the digested peptides is essential for generating high quality mass spectra in LC-MS/MS analysis. The sample preparation methods, such as Filter-Aided Sample Preparation (FASP), acetone precipitation followed by in-solution digestion (AP), strong cation exchange-based centrifugal proteomic reactor (CPR), are commonly used for proteomic sample process, but the additives removal efficiency and the application scope of these methods need to be further investigated. In this study, we demonstrated an integrative work flow for systematical small molecular additives quantification as well as peptide/protein identification to provide a comprehensive evaluation for additive cleanup effect of proteomic sample preparation pipelines. The multiple reaction monitoring (MRM)-based LC-MS approach was developed for six additives (i.e., Tris, Urea, CHAPS, SDS, SDC and Triton X-100) quantification. For the peptide and protein identification, although FASP outperformed the other two methods, these three methods were complementary to each other, in terms of peptide/ protein identification, as well as GRAVY index and amino acids distributions. By integrating analysis of small molecular quantification and protein identification datasets, we first bridged the quantitative influence of additive concentration to the peptide analysis performance. Our results provide a valuable dataset for appropriate sample preparation method selection and a guideline for evaluation of small molecular additives cleanup efficiency in the sample preparation procedures.