Improved sensitivity proteomics by postharvest alkylation and radioactive labeling of proteins

We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [H-3]iodoacetami de or I-125. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkyla ting reagents can increase the number of proteins able to be detected by io dination chemistry. Proteins were alkylated with beta-(4-hydroxyphenyl)ethy l iodoacetamide, or with 1,5-I-AEDANS (the Hudson Weber reagent). Subsequen t iodination using the lodo-Gen system was found to be most efficient. The enhanced sensitivity obtainable by using these approaches is expected to be sufficient for visualization of the lowest copy number proteins from human cells, such as from clinical samples. However, we argue that significantly improved methods of-protein separation will be necessary to resolve the la rge number of proteins expected to be detectable with this sensitivity.