Lys-N: A versatile enzyme for proteomics

Abstract

To overcome the difficulties of analyzing proteins in highly complex samples an improvement in proteomics strategies is needed. The combination of multiple proteases, peptide separation and fragmentation techniques may reduce sample complexity and improve the analysis of different sub-groups of peptides, including low abundant proteins and peptides. In this thesis, I introduce a relatively new protease in proteomics workflows and demonstrate the strength of combining its proteolytic peptides with multi-dimensional separation techniques and different fragmentation techniques, to decrease sample complexity and to improve sample identification. In chapter 2, we evaluate the fragmentation pattern observed for peptides generated by the metalloendopeptidase Lys-N using electron transfer dissociation (ETD). The enzyme Lys-N generates peptides with a lysine residue at the N-terminal. We show that ETD sequencing of BSA generated peptides with an N-terminal lysine, and no other basic residue in the sequence, result in spectra dominated by c-type fragment ions. To confirm the result, fragment ion statistics were increased by analyzing Lys-N generated peptides from a cell lysate. Additionally, we show that these doubly charged Lys-N peptides containing a single lysine at the N-terminal can be selectively enriched for by using low-pH strong cation exchange (SCX). In chapter 3, we further evaluate the SCX based fractionation of peptides generated from the metalloendopeptidase Lys-N. Here, we interestingly show that it is possible to obtain fractionation profiles where different subgroups of Lys-N generated peptides such as, acetylated N-terminal peptides, singly phosphorylated peptides, peptides with a single basic residue and peptides with multiple basic residues can be separated. We demonstrate that the combination of Lys-N digestion, low-pH SCX and reversed phase (RP) separation, with CID and ETD induced fragmentation, is a powerful approach for global proteome and phosphoproteome analysis. In chapter 4, the metalloendopeptidase was explored for its use in MALDI-MS/MS proteomics applications. Lys-N generated peptides from a BSA digest were analyzed by MALDI-MS/MS, which resulted in simple and straightforward CID spectra, containing complete b-ion series. Statistical analysis was again performed to confirm the results where a cell lysate was digested to obtain a higher number of doubly charged Lys-N peptides. Last, it was found that the simple straightforward MALDI CID spectra can be used to facilitate de novo sequencing. In chapter 5, the proteolytic performance of the metalloendopeptidase Lys-N was evaluated. As a model system BSA was used to validate the performance of Lys-N when using a number of classical proteomics sample handling conditions. We demonstrate that Lys-N has many interesting and useful characteristics as it was found to be highly thermo-stable and to have a high tolerance towards certain denaturing agents, such as urea and acetonitrile. Furthermore, it was found that by increasing the digestion temperature a decrease in incubation time could be achieved. Additionally, we demonstrate that Lys-N is able to cleave adjacent to single-methylated lysines and partially adjacent to di-methylated lysines, which may be useful when analyzing naturally occurring post-translational modifications