## 3 Results and Discussion
### Intact Mass Analysis Can Guide Small-Scale Test Expression of Multiple Clones  
The above setup and throughput enable us to extend MS analysis to an earlier stage in the protein production process, namely the parallel small-scale expression testing of multiple expression clones. This is routinely performed in a 96-well format, which will cover \(\sim\)5-10 target proteins each sampling 8-12 different N- and C-terminal truncated variants, as part of our multi-construct approach to optimize recombinant expression and solubility [1,4]. This analysis poses additional challenges as the proteins are only partially purified (after one step of affinity purification), the yields are variable and may be limiting, and the proteins are eluted in a complex solution with a high concentration of imidazole. However, where possible, such early-stage analysis can provide valuable information on the selection of constructs before embarking on large-scale purifications. An example of summary intact mass analysis for a subset of constructs from a 96-well test expression is shown in Table 1 (the full dataset can be found in Supplementary Material Section 5, along with the complete SDS-PAGE analysis in Section 6). LC-MS acquisition time was 5.6 h (overnight) and data analysis time was approximately 3 h. Spectra were obtained for constructs which were scored as high or medium expressers by Coomassieie Blue staining following SDS-PAGE analysis (Figure 2 dilutions). Low expressers or non-homogeneous samples did not produce protein spectra therefore bands from these elutions were submitted for MSMS analysis (see Section 3.4). Deviation from the expected theoretical mass was less than 1 Da and as small as 0.02 Da. This degree of mass accuracy was sufficient to confirm correct target protein expression, correct construct expression and that no non-isobaric mutations were present. Subsidiary peaks were always observed which corresponded to multiple sodium adducts (+22 Da). Subsidiary peaks which did not conform to this mass deviation were accounted for as PTMs, such as +178 Da (gluconylation [13]) which are known to occur in the expression system used. In some instances, no peak corresponded to the expected unmodified mass, but could be accounted for by single or multiple PTMs where 100% modification had occurred. Figure 2: SDS-PAGE analysis of both the IMAC elutions (15 \(\mu\)L) and total cell lysates (3 \(\mu\)L) for a subset of the 96-well test expression. These samples include examples of high (F10), medium (E6) and low (F3) expressers. \begin{table}
\begin{tabular}{l l l l l l l l l l l l l} \hline \hline  & \multicolumn{4}{c}{Sample Description} & \multicolumn{4}{c}{**Inited Mass Analysis**} & \multicolumn{2}{c}{**MSMS Analysis N Ention**} & \multicolumn{2}{c}{**MSMS Analysis N Total Protein**} \\ \cline{2-11} \multicolumn{1}{c}{**Constrated**} & \multicolumn{1}{c}{ \begin{tabular}{} **N or C** \\ **Terminal** \\ **Tag** \\ \end{tabular}  } & \multicolumn{1}{c}{ \begin{tabular}{} **Elation Yield** \\ **(SDS-PAGE)** \\ \end{tabular}  } & \multicolumn{1}{c}{ \begin{tabular}{} **Flate Well** \\ **Plate Well** \\ **(SDS-PAGE)** \\ \end{tabular}  } & \multicolumn{1}{c}{ \begin{tabular}{} **Expected** \\ **Mass (Da)** \\ \end{tabular}  } & \multicolumn{1}{c}{ \begin{tabular}{} **Observed** \\ **Mass (Da)** \\ \end{tabular}  } & \multicolumn{1}{c}{\ \begin{tabular}{} **Delta Mass** \\ **Mass (Da)** \\ \end{tabular}  } & \multicolumn{1}{c}{ \begin{tabular}{} **Relative** \\ **Intensity \(\times\)19** \\ \end{tabular}  } & \multicolumn{1}{c}{**Comments**} & \multicolumn{1}{c}{**MOWSE Score**} & \multicolumn{1}{c}{**Identity**} & \multicolumn{1}{c}{**MOWSE Score**} & \multicolumn{1}{c}{**Identity**} \\ \cline{2-11} \  
Intact mass analysis of integral membrane proteins would be highly desirable, especially since their size cannot be reliably estimated by SDS-PAGE. The literature on MS characterization of IMPs is substantial (reviewed by [PERSON] [14]), yet intact mass analysis of these proteins is not routine.