Isotope-Dilution Mass Spec
Based on recent developments in quantitative mass spectrometry, biological reference materials have been generated that can be used as internal (quality) controls for proteomics utilizing the characteristics of isotope-dilution mass spectrometry (ID-MS).
ID-MS is recognized as one of the best quantitative technologies in bioanalytics. In the last few years, ID-MS has found widespread application in proteomics. Usually synthetic or recombinant proteins are used as isotopic standards and spiked into samples to serve as internal standards for quantification. Currently standards must be generated one by one by incorporating isotope-labeled amino acids into the peptide sequence by chemical synthesis or by addition to culture media.
Conversely, chemical labeling, attaching small tags that carry isotope differences to the analytes, presents a simpler approach without the need to actually synthesize any peptides. In general, two different methods of chemical labeling are used—isotopic mass labeling and isobaric mass labeling.
Isotopic mass labeling leads to a distinct mass difference of structurally identical reference and target analyte. The ratio between the heavy and light analyte are used for quantification. Isobaric mass labeling leads to isobaric analytes that can be differentiated by isotopic mass reporters released from the analyte during tandem mass spec. Again, the ratio between different mass reporter intensities is used for quantification
For both modes, absolute quantification is achieved by spiking in the reference in known amounts. Otherwise, relative quantification values are obtained.
The Tandem Mass Tag® (TMT®) core structure invented by Proteome Sciences allows for the generation of different sets of isotopic and isobaric mass tags. TMTduplex tags incorporate one 13C each, giving an isobaric set of two tags (Figure). TMTsixplex tags incorporate four 13C and one 15N, giving an isobaric set of six tags. Combining TMTzero with either TMTduplex or TMTsixplex gives a set of isotopic tags, as would a combination of TMTduplex with TMTsixplex.
The principle behind chemical labeling is that the quantitative ratio between individual proteins from different samples remains constant after labeled samples are mixed. This conservation is the key for subsequent multistep separation and analysis, providing much better control, precision, and accuracy.
Mixing of labeled samples should be performed as early as possible during any workflow, and one of the samples needs to be a common reference sample
The most commonly applied labeling method uses the reaction between protein amine functions and succinimide esters, resulting in labeling of >98% of amine functionalities. As several amine functionalities (amino-terminal amino acids and epsilon-amine functions of lysines) are present in almost all (human) proteins, virtually the entire proteome is accessible to mass spectrometric quantification with this technology.