Enhancing the mass spectrometric identification of membrane proteins by combining chemical and enzymatic digestion methods

Abstract

Membrane proteins are critical for many cellular events, including cell signaling, molecular transport, and extracellular interactions. One third of the genome is estimated to encode membrane proteins, which are correlated with disease progression and can serve as promising biomarkers and drug targets. Modern mass spectrometry (MS)-based proteomics techniques facilitate the global analysis of proteins in complex biological samples; however, the hydrophobicity of membrane proteins inhibits their comprehensive analysis. Since membrane proteins are not easily accessible by proteases in aqueous solutions, a combinatorial method incorporating chemical and enzymatic digestion is presented here to improve the digestion efficiency of membrane proteins for MS analysis. Chemical digestion with 2-nitro-5-thiocyanatobenzoic acid (NTCB) was supplemented with enzymatic digestion (Glu-C, or Lys-C and trypsin) to determine the optimal combination of digestion methods. Three parallel experiments were performed with membrane protein extracts from HEK293T cells, and the results demonstrated that combining NTCB with Lys-C and trypsin resulted in the greatest number of identified peptides and proteins while the least number of peptides and proteins were identified by using sequential digestion with NTCB and Glu-C. By integrating chemical digestion before enzymatic digestion, NTCB could more easily access cleavage sites within membrane proteins, and the resulting peptide fragments were thus more accessible by proteases. The combination of chemical and enzymatic digestion presented here proved to be effective for membrane protein analysis.