Improved sensitivity proteomics by postharvest alkylation and radioactive labelling of proteins.
We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [3H]iodoacetamide or 125I. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkylating reagents can increase the number of proteins able to be detected by iodination chemistry. Proteins were alkylated with beta-(4-hydroxyphenyl)ethyl iodoacetamide, or with 1,5-l-AEDANS (the Hudson Weber reagent). Subsequent iodination using the Iodo-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 large number of proteins expected to be detectable with this sensitivity.
Vuong
G L
GL
Institute for Cell Biology, Department of Molecular Biology, University of Tübingen, Germany.
Weiss
S M
SM
Kammer
W
W
Priemer
M
M
Vingron
M
M
Nordheim
A
A
Cahill
M A
MA
D000081:Acetamides; D000478:Alkylation; D002700:Chloramines; D003545:Cysteine / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005240:Feasibility Studies; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D006997:Hypochlorous Acid; D007457:Iodine Radioisotopes / Q000032:analysis*; D007525:Isoelectric Focusing; D007553:Isotope Labeling / Q000379:methods*; D008223:Lymphoma / Q000378:metabolism / Q000473:pathology; D009281:Naphthalenes; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D016877:Oxidants; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D020543:Proteome; D012680:Sensitivity and Specificity; D016622:Silver Staining; D013451:Sulfonic Acids; D014105:Tosyl Compounds; D014316:Tritium / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000737:chemistry
C418302:(4-hydroxyphenyl)ethyl iodoacetamide; D000081:Acetamides; D002700:Chloramines; D007457:Iodine Radioisotopes; C418282:N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid; D009281:Naphthalenes; D009363:Neoplasm Proteins; D016877:Oxidants; D011506:Proteins; D020543:Proteome; D013451:Sulfonic Acids; D014105:Tosyl Compounds; C016300:chloramine-T; D014316:Tritium; D006997:Hypochlorous Acid; D003545:Cysteine