Examining the variables which influence strong cation exchange of peptides, and their implications on the first dimension of separation in liquid chromatography tandem mass spectrometry

Abstract

Orthogonal separations are often employed to improve the capacity of mass spectrometry for proteome analysis. The gold standard of orthogonal peptide-level separation utilises strong cation exchange (SCX) coupled to reversed phase chromatography in an online format. However, the gradient conditions used in SCX fail to account for maximal sample recovery, nor for optimising the degree of fractionation; most SCX separations primarily rely on salt gradients, rather than employing pH ramps to recover peptides. Additionally, online separations must compromise the types of solvents used to reduce matrix effects experienced in electrospray ionisation mass spectrometry. This thesis endeavours to understand the conditions governing retention and recovery of peptides from SCX, primarily pH and ionic strength, using two novel offline approaches. The ProTrap XG presented in Chapter 2 is a centrifugal column chromatography device which allows for multiple simultaneous separations, with an almost unlimited choice in separation buffers. Using this system, it was determined that altering the pH of the eluting buffer was the primary variable impacting peptide retention on the SCX column, while ionic strength played a minor role in the separations. A pH gradient in the absence of salt was optimised, loading peptides at pH 2.1 and eluting between pH of 3 and 11, with an average recovery of 80  11% and 2600  800 E. coli peptides identified per replicate. Attempts at optimising the E. coli peptide separation through combining pH and salt resulted in 90% recovery. However, the device had challenges in reproducibility due to variable flow rates and spin times, which arose due to variations in column consistency. This is addressed in chapter 3 with a novel automated HPLC-based separation platform capable of multiple solvent deliveries for optimised SCX separation. The Strong Cation Exchange Clean-up Liquid Chromatography (S-CLC) system separates yeast peptides and desalts the fractions and measures recovery by LC-UV. Two key components distinguish this system from traditional multidimensional chromatography systems, namely two distinct pumps are employed (one to load/ elute on SCX and the other to load/ elute from reversed phase). The second is to use an autosampler to inject the solvents used to recover peptides from SCX, which like the ProTrap XG, allows tailored selection of buffers to optimise separation in automated fashion. Separation by S-CLC confirmed that pH is the primary variable in altering the retention of peptides in SCX, while salt gradients provide counterions that compete for interaction with the column. Salt and pH were combined to elicit the removal of peptides from the SCX column and compared to the gold standard method. While both methods identified a similar number of peptides the MuDPIT method had higher levels of overlap between fractions, while the combined salt and pH gradient had decreasing overlap. Based on these results, an optimised separation on SCX should be developed using univariate separations with pH in the absence of salt, and a salt gradient under constant pH conditions. Once this is complete, salt and pH gradients can be combined until obtaining even peptide partitioning. On a high-resolution mass spectrometer, it is expected that there will be a significant increase in the number of identified proteins when compared to traditional proteomic separation methods.