## 3 Results and Discussion
### 3.3. Intact Mass Analysis Is Used to Reveal Expected/Unexpected Post-Translational Modifications  
PTM characterization by pin-pointing the location of these modifications within a construct sequence is not a high throughput technique. Nor is it trivial with the limited material available from small scale test expression. Since speed is critical to high throughput sample preparation, acquisition and data analysis, no attempt is made to do this. Relying instead on intact mass measurement alone, the presence or absence of all commonly observed PTMs may be reliably and routinely detected. An intact mass spectrum from a test expression showing typical PTMs is shown in Figure 3. Protein intact mass spectra from high expressers typically show protein peaks with signal to noise of greater than 200:1 and mass accuracy better than \(\pm\)1 Da. This degree of mass accuracy allows subsidiary peaks such as sodium adducts and PTMs to be readily distinguished. Although Unimod [12] and DeltaMass [22] websites provide extensive lists of known protein mass variants, caution must be used in assigning these to the target protein because nearly all these mass variants are extremely rare, and many are chemical or isotopic derivatives, not possible outside a specific experimental context. More useful is a knowledge of which PTMs are known to occur in the expression system being used and with what frequency. Table 2 lists 19 PTMs observed by us, with a further eight mass differences with their likely interpretation. In some instances, verification of these mass differences is possible. Phosphorylation, for example, can be verified by an additional intact mass measurement following enzymatic removal of phosphate groups (e.g., lambda phosphatase) [23; 24; 25]. N-terminal modifications will be lost following TEV cleavage of an N-terminal tag. Histidine tag-associated modifications such as gluconoylation will also be lost following TEV cleavage. Intact mass measurements can be used to monitor deliberate modifications of the protein, such as biotinylation [26], reductive methylation [27], glycosylation [28], incorporation of SeMet or isotopically-labelled amino acids, or the removal of the tag by TEV cleavage. In all cases where a mass discrepancy cannot be unequivocally accounted for, the expression clone is (re)-sequenced. Peaks with double the calculated mass, require careful interpretation. The denaturing LC-MS conditions described do not always result in total denaturation. Some structure can still be retained and consequently, though rarely, dimers and other structures may be observed. More commonly, maximum entropy deconvolution may generate artifactual peaks of double or half the protein mass. In this case, the sodium adducts normally associated with these peaks will appear to have double or half the proper sodium mass shift (+44 Da or +11 Da). So called top-down MSMS techniques [29] are now available for protein identification and mapping of post translational modifications using the latest generation of high end mass spectrometers. However, it should be borne in mind that no MSMS technique generates full sequence coverage; hence, the possibility that PTMs will be missed. In contrast, intact mass measurement allows detects the presence of all PTMs wherever their location in the sequence, allowing confirmation of the entire covalent structure. A commonly-used alternative to ESI-TOF is Matrix-Assisted Laser Desorption/Ionization (MALDI)-MS. Accurate mass determination of short peptides by MALDI compares favorably with ESI. However, the sensitivity, resolution and mass accuracy of MALDI instruments decreases rapidly for larger analytes such as intact proteins, such that accurate mass measurement of proteins greater than 15 kDa is generally not possible. While ESI protein mass measurements have a typical accuracy of \(\pm\)10 ppm, similar measurements by MADLI have an accuracy of \(\pm\)1000 ppm; hence, the information gained is of limited value and unsuitable for construct and PTM identification.