Protein and peptide analysis using liquid chromatography/mass spectrometry (LC/MS) applications can be challenging, time consuming, and/or expensive using traditional techniques for sample preparation. For example, challenges occur when sample preparation or analysis require multiple steps, since each additional step can add additional time to the sample preparation and potentially introduce errors to the analysis. Additional steps can also result in a loss of product, i.e., the peptide or protein of interest. This loss can occur, for example, during each transfer of the solution containing the protein or peptide prior to LC and/or mass spectrometric analysis, thereby resulting in decreased sensitivity/resolution.
Though denaturing a peptide or protein can improve mass spectral detection as a result of increased charging and exposure of ionisable groups (e.g., cleaving the disulfide bridges in a peptide or protein, followed by an alkylation step to ensure that the reduced disulfide bonds will not reform), such a reduction of disulfide bonds typically require an incubation step, which can decrease throughput and increase the complexity of sample preparation. Furthermore, incubation conditions can lead to alteration of side chain residue of protein and peptides, thus further complicating the analysis and interpretation. For example, while the length of the incubation period depends on reducing agent used, a 30 to a 60 minute time period is typically required and controlled conditions (e.g., pH) need to be carefully considered to avoid side reactions. Moreover, the incubation period is typically followed by the addition of alkylating agent to prevent the reformation of the disulfide bonds, which can lead to disulfide bond scrambling. Iodoacetamide, for example, is commonly added after reduction of the peptide, but is light sensitive and typically adds an additional 30 minutes of reaction time to the sample preparation process.
Accordingly, there remains a need for improved methods and systems for the reduction of peptides and proteins for their mass spectrometric analysis.