An optimised method to isotopically label pure synthetic peptides ‘in‐house’ for absolute quantification in bottom‐up proteomics

Rationale Heavy‐labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)‐based bottom‐up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. Methods We describe an efficient, low‐cost optimised method to enable ‘in‐house’ heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC‐MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin‐catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. Results Step‐by‐step instructions are provided on how to execute this protocol with high‐throughput adaptations utilising a 96‐well plate and a liquid‐handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. Conclusions The application of the ‘in‐house’ labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.