## 3 Results and Discussion
### MS Is Complementary to SDS-PAGE in a Structural Biologist's Toolkit  
The value of intact mass and MSMS analyses is that information is gained over and above that from SDS-PAGE at minimal cost in terms of time and money. Foremost, it can confirm that the over-expressed protein is indeed the target. For low to medium expressers as judged by SDS-PAGE, one might expect these constructs would not normally be pursued for scale up. However, MSMS confirmation of target protein, for even some of these constructs, would help guide the next round of construct boundary optimization. Additional information from high/medium expressers is confirmation that the observed mass matches the theoretical mass of the construct. We assume from this that the construct is correct, the protein sequence is correct and that at this stage construct DNA sequencing is not prioritized. If present, PTMs are evident in the intact mass spectrum. They fall into two categories: N-terminal processing, which may or may not involve the affinity tag, and modifications which may affect protein activity or the likelihood of crystallization such as phosphorylation or glycosylation. Intact mass spectrometry is uniquely capable of assessing sample heterogeneity that is not discernible by SDS-PAGE, such as a partial proteolysis of a few amino acids or multiple phosphorylation states.  
The two techniques should be considered as complementary within the structural biology pipeline. Gel analysis is superior to LC-MS for visualization of proteins in complex mixtures, large proteins, complexes and IMPs. In our laboratory, LC-MS analysis is performed on IMAC elutions following SDS-PAGE analysis, using surplus material which would otherwise be discarded. Where intact mass analysis has been unsuccessful and gel analysis indicates that the construct protein has been expressed, in-gel digestion and LC-MSMS analysis determines protein identification. Although less informative than intact mass analysis, LC-MSMS is far more sensitive and is applicable to heavily or unusually modified proteins, truncated proteins, very large proteins and IMPs [32]. Once again, it is done using surplus material which would otherwise be discarded. While this information is clearly useful, it will not actually be used in a structural genomics pipeline unless it is available quickly. Using the method described here, intact mass data for a 96-well test expression plate can be available the following day. A  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Expression System** & **Contaminant Protein** \\ \hline  & 40S ribosomal protein S16 \\  & 40S ribosomal protein S3 \\  & 40S ribosomal protein S3a \\  & Eukaryotic translation initiation factor 3 subunit A \\  & Guanine nucleotide-binding protein subunit beta-like protein \\  & Host origin & Heat shock 70 kDa protein cognate 4 \\  & Tubulin alpha-1 chain \\ Insect cell & Tubulin beta-1 chain \\  & Histone H2A \\  & Histone H2B \\  & Histone H4 \\ \hline  & Early 39 kDa protein \\ Viral origin & Major capsid protein \\  & Probable endochitinase \\ \hline \hline \end{tabular}
\end{table}
Table 3: _Cont._complete MSMS dataset can be available in less than 60 h. For soluble proteins, partial datasets available in around 30 h are sufficient when intact mass data constitutes the remainder. Technical discussion of the high-throughput in-gel digestion, LC-MSMS and data analysis methods is provided in the Supplementary Material.