In order to eliminate the lysosomal proteins contaminated in the mitochondrial fraction, mitochondrial suspensions were pre-incubated with lysosomotropic detertgent (100 µM sphingosine, Sigma), 1% Triton X-100 or non-detergent at 37°C for 15 min. After incubation, these mitochondrial suspensions were treated with 0.2, 2, or 20 µg/ml proteinase K for 20 min on ice.
With this method, histones are the predominant proteins and contaminating nuclear proteins are reduced.
The cohort of proteins modified by MitoSNO contrasts with those affected by incubation of mitochondria with the non-targeted S-nitrosating agent SNAP [ 9], which were predominantly outer membrane proteins or contaminating cytosolic proteins.
Further, the purified mitochondria were incubated with trypsin and/or Triton X-100, as trypsin only digests outer membrane proteins and contaminating cytosolic proteins due to mitochondrial membrane protection which is disrupted by the presence of Triton X-100.
Clearly, on the CBB-stained SDS-PAGE, a single major protein band (with low background) was found in the eluted fraction following the sortase self-cleavage of the biotin-tagged srtA fusion protein; in contrast, contaminating proteins were found along with the major protein band in the eluted fraction of the self-cleaved His-tagged srtA fusion protein.
Protein precipitation is not only an effective way of separating proteins from contaminating small molecules but also serves as a preconcentration step for proteins from dilute solutions.
Three major limitations are usually considered in the interpretation of functional proteomics datasets - (i) fidelity of the protein identification; (ii) proteins missed; and (iii) "contaminating" proteins found.
Proteins and contaminating RNA were digested by incubation in the presence of 100 µg/ml proteinase K and 100 µg/ml RNAse A at 45°C for 2 hours.
Introduction of a second purification step with Protein L successfully removed contaminating proteins and heavy chain fragments.
Using HRMS to analyze posttranslational modification has obvious advantages such as low protein quantity requirement, free of contaminating proteins in samples and reading accuracy.
