## 1 Introduction  
Worldwide efforts in structural genomics have led to the development of highly parallel methods of cloning, protein production and crystallization [1, 2, 3, 4]. These technologies are also useful outside the context of structural genomics, in more focused structural projects and in research groups and facilities specializing in protein production. A common feature is the handling of diverse entities (clones, proteins) in parallel, and there is an important need for robust and informative measures to assess the quality and properties of the final purified protein. No less important is the ability to achieve a comparative assessment of different clones or methodologies, in order to select the best route to obtain the target protein. In laboratories where it is available, mass spectrometry (MS) is used to confirm the identity, purity and structural homogeneity of purified proteins or to characterize post-translational modifications (PTMs) prior to entering crystallization trials [5]. Whilst the use of MS purely as a quality control tool is prudent, an important benefit of applying MS to the structural genomics pipeline lies in influencing the experimental process in real time. However, typical turnaround times of days to weeks usually mean that the analysis is after the fact, with little chance of influencing the flow of an experiment in real time. Figure 1 describes schematically our protein production and analysis pipeline and indicates the points of use and the impact of MS in the process. We have applied high-throughput methods for intact mass analysis and in-gel tryptic digest MSMS analysis to the early, small scale (1-3 mL) test expression phase of our pipeline. Intact mass analysis can confirm expression of the target protein with the expected sequence, identify PTMs based on assignment  
Figure 1: Integration of mass spectrometry (MS) in the protein production pipeline. of mass differences and quantify expression yield. This data is additional and complementary to SDS-PAGE analysis, allowing informed decision making, prior to scale-up. It is also complementary to DNA sequence data, in that it identifies features of the protein product, as well as detecting post-cloning effects including sequence changes and clone cross-contamination. In fact, in a high-volume operation where most of the constructs do not yield soluble protein, we have found that in-house MS is more practical than sequencing of hundreds of DNA constructs. In intact mass analysis where the discrepancy between observed and expected mass is too large to be accounted for by modification, or where the target protein is not readily amenable to intact mass measurement (if, for example the target is larger than 80 kDa or is an IMP), tryptic digest MSMS analysis will confirm the identity of the target protein [6]. Although less informative than intact mass, most proteins are amenable to this analysis. IMPs express at lower abundance, have anomalous SDS-PAGE mobility and poor gel resolution, compounded by glycosylation when this is present [7]. Together, these factors render SDS-PAGE alone a particularly unreliable method for IMP identification following test expression. Western blots may be used when suitable antibodies are available; otherwise MSMS analysis is our method of choice. Rather than focusing on in-depth protein analysis, which is incompatible with high throughput and fast sample turnaround, these improvements allow high quality MS data to be generated quickly. Taken together, these methods allow low cost, high-throughput protein characterization of 96-well test expressions with rapid turnaround. We show typical data generated with these methods and we argue that the timely availability of MS protein characterization data at the earliest test expression stage of a structural genomics pipeline improves the efficiency and cost effectiveness of the entire pipeline.