Lysine-directed staining of proteins for MS-based analyses.

Visualization of proteins and MS-based analyses are elemental tasks in modern biochemistry. Nevertheless, reports about covalent protein dyes and their suitability for subsequent MS experiments remain scarce. In a recent work, we demonstrated that covalent prestaining of proteins with Uniblue A drastically speeds up proteomic workflows. The present study introduces dabsyl chloride as another truly MS-compatible protein stain. Remarkably, although Uniblue A and dabsyl chloride employ different nucleophilic reaction mechanisms, both are highly specific for lysine residues. The predictable peptide modifications allow easy integration into state-of-the-art bioinformatic workflows. Further, lysine-directed derivatizations with hydrophobic reagents such as dabsyl chloride complement the cysteine-directed ALiPHAT strategy for increasing the sensitivity of peptide identifications.

Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A.

BACKGROUND: The identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools. CONCLUSIONS AND SIGNIFICANCE: The Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.