Abstract:

Facing the needs of multidimensional and multi-sample proteome quantification in the field of biology and precision medicine, high-throughput quantitative labeling and analysis have become the trend in recent years. A novel cross-sequence labeling equipment for high-throughput proteome based on pseudo-isobaric dimethyl labeling (pIDL-StageTip) was established. The equipment needs only simple devices and centrifugal force. The throughput of quantitative labeling is effectively increased, and the controllability of the two-step labeling reaction time at the terminals of the peptides and the ease of operation are ensured. By optimizing the labeling conditions of NaBD₃CN and NaBH₃CN under acidic conditions respectively, labeling efficiency of the standard proteolysis achieved 100% and labeling selectivity was more than 95%. Even in the complex human proteome, the labeling efficiency was greater than 99% and the labeling selectivity was 100%. The quantitative method based on the equipment showed high quantitative accuracy and precision. The equipment provides a reliable solution for quantitative proteome labeling with high operability, accuracy, and throughput.

Key words: cross-sequence labeling using site-selectivity, high throughput, proteome labeling equipment, pseudo-isobaric dimethyl labeling (pIDL)