An Intelligent Data Acquisition Approach to Increase the Throughput of Multiplexed Proteome Analyses

Isobaric labeling enables the multiplexed analysis of up to 18 proteomes or PTMomes in a single analysis and provides information on protein and peptide abundance across all samples simultaneously. For certain applications, reactive cysteine profiling and single cell proteomics, we sought to improve the throughput of analyses to 36-plex by employing a hyperplexing approach that combined both isobaric labels with additional mass offsets through light/heavy cysteine probes or SILAC. Previous implementations of hyperplexing have been challenged by the selection and quantification of both peptides within a hyperplexed pair, leading to some peptides/proteins only being quantified in 18 of the 36 samples. To ensure quantification of both peptide pairs we introduce PairQuant an intelligent data acquisition approach implemented through inSeqAPI, our own instrument API program. PairQuant sequences peptides within hyperplexed pairs in real time and only triggers quantification scans after both peptides within a hyperplexed pair have been identified. Furthermore, if the instrument does not select both peptides within a hyperplexed pair for MS/MS, PairQuant will send PairSeq scans in an attempt to sequence the missing peptide. We use pair quant for 36-plex cysteine profiling analyses as well as 28-pelx single cell analysis and demonstrate an increase in the number of hyperplexed pairs where each partner peptide was quantified.