Figure 2 A general scheme for protein identification utilizing an MS-based approach applied in the authors’ laboratory.

doi:10.1201/9780203910696-18

Chapter

Figure 2 A general scheme for protein identification utilizing an MS-based approach applied in the authors’ laboratory.

ABSTRACT

Using the above protocol, a scientist can comfortably digest and extract 25 samples in less than 36 hr, which limits throughput to approximately 100 in-gel digests and extractions per week. (In our experience, significant human error can be introduced over time while manually processing more than 25 samples per day.) Reducing the digest time from overnight to 2-3 hr can improve the throughput, but not significantly. However, a 2D gel of a cell lysate can contain literally thousands of unique proteins, seen as stained spots. Even if the spots are excised from the gel manually, hundreds of samples requiring digestion and extraction can be generated per day. Setting aside the fact that in a proteomics project it is rare that each experiment would require every single spot from the gel to be identified, it is obvious that many more samples can be generated per week per scientist than digested and extracted manually.