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We set out to develop an easy-to-use system for in vitro synthesis of recombinant eukaryotic proteins containing posttranslational modifications. A wide range of eukaryotic proteins require such modifications (e.g., phosphorylation, glycosylation, signal peptide cleavage) for correct folding or to display full functional activity.

Optimized sample preparation is crucial for successful and sensitive detection of peptides by MALDI-TOF MS. In particular, the protein identification of low femtomole amounts of analyte requires further clean-up and concentrating steps to acquire significant spectra. This aspect is even more pronounced if the analysis of post-translational modifications is the goal. A prominent example is the mapping of protein phosphorylation sites, which is often hampered by the lack of phosphopeptides in spectra of crude digests. For that reason, very often micro-columns filled with reversed-phase material for clean-up, or IMAC or TiO2 resins for phosphopeptide enrichment, are employed prior to MALDI-MS analysis.

Proteomic profiling of biological samples using mass spectrometry has been a goal for biomarker discovery and disease research for some time. It remains the current challenge to reliably and reproducibly isolate and to sequence the potential biomarkers found in high-throughput screening efforts by tandem mass spectrometry (MS/MS) to differentiate the important and meaningful biomarkers from ambiguous pattern artifacts. To this end, top-down and bottom-up proteomic workflows have yielded advantages and disadvantages, depending upon implementation into the research field.