The use of underloaded C18 solid-phase extraction plates increases reproducibility of analysis of tryptic peptides from unfractionated human plasma

Bottom-up proteomics requires the digestion of proteins into peptides by processes that use salts for denaturing and buffering purposes. These salts need to be removed prior to mass spectrometry analysis to reduce ion suppression; solid-phase extraction (SPE) is a commonly used strategy. There are many commercially available SPE sorbent types and sizes, which are generally provided with manufacturer recommendations for use, including protein loading capacity. We found that these general suggestions were often not ideal, and our data suggest that context-specific evaluation of sorbent type and amount can improve reproducibility. Specifically, the universal Oasis HLB sorbent provided better retention of the more hydrophilic peptides than the traditional C18 reversed-phase SPE, but it did so at the expense of an increased loss of the more hydrophobic peptides. We found that increasing the amount of the C18 sorbent beyond the manufacturer’s guidelines decreased breakthrough (i.e., increased retention) of 12 hydrophilic, identifiable peptides without loss of hydrophobic peptides. This procedure was robust in a 96-well plate format.