Universal toolset for mass spectrometric analysis of intracellular peptidome and small protein fraction

Abstract

The analysis of native intracellular peptidome has gained significant attention in recent years. However, there is still a need for more knowledge regarding various sample preparation methods that facilitate efficient and reproducible recovery of peptides, which can then be analyzed using quantitative liquid chromatography-mass spectrometry. A similar situation exists in small proteome research, typically defined as polypeptides with masses of less than 100 amino acids, often too long for easy identification without enzymatic digestion. In this context, we describe a set of methods that involve simple denaturation and solid-phase extraction of polypeptides, applicable for isolating short intracellular polypeptides within the desired length range. Our work demonstrates the efficiency and reproducibility of these methods for quantitative analysis of the peptidome in mammalian cells. Additionally, we investigated the flexibility of adjusting the mass range through ultrafiltration. We have shown that these methods can be adapted for highly efficient enrichment and fractionation of small proteins, resulting in polypeptide isolates suitable for tryptic digestion and intact protein analysis. Moreover, we describe the use of freely available computational tools that can effectively manage the analysis of the resulting data. The research presented here will benefit the global scientific community in both fundamental (protein turnover, proteolytic processing, non-canonical open reading frames, etc.) and applied sciences (bioactive/neuro peptide discovery, precision medicine, vaccines, etc.), and other areas that could benefit from selective analysis of short native polypeptides.