Post-translational modifications molecular mass measurement

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Post-translational modifications to proteins are biochemical in origin and alter the measured molecular mass relative to that calculated for an untranslated sequence. 

The protein identification or sequence determination of a protein can be achieved using two different approaches top-down [22, 23] and bottom-up [24], A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25], This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco-... 

Measuring molecular masses to ascertain an identity and to evidence post-translational modifications. 

While simple molecular weight measurements do not provide information on the amino acid sequences of peptides, they have been profoundly useful for verifying sequences which are inferred from the nucleic acid sequences of the genes encoding the peptide. In particular, they have been used to verify the sequences of peptides produced by recombinant techniques or by total chemical synthesis, or to reveal possible post-translational modifications. More specific information, however, can be obtained by comparative mass mapping of tryptic (or other enzymatic) digests. This approach is particularly useful when the molecular-ion mass exceeds the mass range of the plasma desorption technique. 

In a scenario that occurs often in a protein biochemistry lab setting, a researcher has isolated a protein of unknown function, or he/she has overexpressed a protein in Escherichia coli and wishes to characterize it. A common practice today is to submit a small amount of the protein to a core mass spectrometry laboratory for a molecular weight measurement. Using either ESI or MALDl, a molecular weight with a precision and accuracy of 0.05% or better can be measured. This, of course, depends heavily on the purity of the protein sample, the relative size of the protein, the presence or absence of post-translational modifications (PTM) (e.g., phosphorylation, glycosylation, etc.), the resolution of the mass analyzer, and so on. Primary structure information (i.e., amino acid... 

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