Abstract
A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.
Highlights
A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies
A powerful technique to emerge is the combination of liquid chromatography and mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS)
As powerful and complementary as current genomic and proteomic tools are, they suffer several shortcomings. Tools such as cDNA microarrays are extraordinarily powerful for the simultaneous detection of thousands of gene products, mRNA levels do not necessarily correlate with protein expression levels [15, 16]. 2D PAGE is of limited use in verifying DNA microarray results, because it is difficult to predict in advance which of the potentially thousands of spots corresponds to a given protein because of the spectrum of possible post-translational modifications
Summary
Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. Tools for the measurement and analysis of gene and protein expression patterns are at the core of several recently defined disciplines, functional genomics, transcriptomics, proteomics, and subfields such as pharmacogenomics and pharmacoproteomics Among these tools, differential display of mRNA is performed routinely using cDNA microarrays [1, 2].
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