## 3 Results and Discussion
### MSMS Data Is Used to Confirm Identity of Target Proteins  
A second level of investigation after intact mass and SDS-PAGE analyses is the LC-MSMS of excised gel bands, usually performed for a subset of the 96-well constructs where (i) intact mass of a low expresser is not conclusive or there are multiple bands of similar mobility to the target protein in SDS-PAGE. Analysis of a full 96-well plate would take 60 h, but in practice, only those bands that have not been confirmed by intact mass analysis need be submitted. Typically, the turnaround time is therefore less than 48 h. MSMS analysis of a complete 96-well plate excepting IMPs is rarely justified, since  
Figure 3: Intact mass spectrum from nickel elution of test expression SPIN1 (21–262) (theoretical mass 30,189.2 Da) showing four most frequently observed PTMs. Sodium adducts are unlabeled. successful identification based on intact mass analysis obviates the need for MSMS. Results for a batch analysis of gel bands from the corresponding subset of constructs (see Section 3.2) are shown in Table 1 (the full dataset can be found in Supplementary Material Section 5). For example, the identities of samples E06-F07 were not confirmed by intact mass. Expression levels ranged from low to high, but in all cases, target identities were confirmed by MSMS. Additionally, samples E08 and E11 each have an intact mass discrepancy of +2111 Da, while MSMS analysis shows that this is the target protein. Re-sequencing of these clones revealed a frameshift near the C-terminus which was previously overlooked, leading to read-through beyond the intended stop codon. This was remedied by re-cloning. In addition, it can be seen from the gel (Figure 2) that the purified proteins in samples E09, E10, E12 and F01 appear as doublet bands. Samples F03, F04 and F05 show weak levels of expression, requiring further optimization before scale-up. Typical \"hits\" for proteins comprised of MOWSE scores of several hundred, well above the significance threshold for databases of this size. For successful target protein identifications, hits occurred in both construct and Uniprot searches. Uniprot hits only occurred for host contaminant proteins. Table 3 lists commonly expressed proteins observed by MSMS from bacterial and insect expression systems. Peptide coverage for IMPs is lower than for soluble proteins because tryptic cleavage sites are rare in membrane spanning regions and these are less accessible to proteases in general [31]. For IMPs, we therefore compensate with five-fold greater volume digest for LC-MSMS, or if necessary 40-fold. Less stringent criteria for identification of known protein targets are needed than in a proteomics context; hence, a single high scoring peptide match is often sufficient for IMP identification. While both detergent (used for IMP extraction) and high imidazole have a negative impact on intact mass analyses, they do not interfere with either SDS-PAGE purification or subsequent tryptic digestion and LC-MSMS. Similarly, partially degraded proteins or those which have aggregated and are insoluble are readily amenable to in-gel digestion and LC-MSMS. Figure 2 also includes analyses of total protein from IMAC purification. We do not routinely analyze this material, but here it is instructive to see that very high levels of expression are observed for nearly all constructs. The barrier to successful purification appears to be protein solubility, something which must be borne in mind during construct design. \begin{table}
\begin{tabular}{c c} \hline
**Expression System** & **Contaminant Protein** \\ \hline \multicolumn{3}{c}{50S ribosomal protein L2} \\ \multicolumn{3}{c}{60 kDa chaperonin} \\ \multicolumn{3}{c}{Bifunctional polymyxin resistance protein ArnA} \\ _E.coli_ & Catabolite gene activator \\ \multicolumn{3}{c}{Chaperone protein httpG} \\ \multicolumn{3}{c}{Chloramphenicol acetyltransferase} \\ \multicolumn{3}{c}{Triosephosphate isomerase} \\ \hline \end{tabular}
\end{table}