Dear Colleagues,
I would like to seek your advice on another question:
Lately, Our lab bought a commercial enzyme, and somehow we've run it on a SDS-PAGE gel. Intriguingly, we observed additionally 3-4 distinct bands (of much lower molecular weights).
MALDI-TOF/TOF protein identification of these extra bands all matched the commercial enzyme. Nevertheless, in certain samples, few peptide peaks were observed in the PMF. As a result of this, albeit the Mascot result showed a statistically significant match, the overall Protein Score as well as the PMF sequence coverage were likewise relatively low (for such certain samples). Based on the above information, May I inquire:
1. Could I conclude that all the extra bands are indeed 'fragments' of the commercial enzyme? Have you encountered similar scenarios before?
2. The protein bands were MS-compatible Silver-stained. Some seemingly 'intense' bands, however, produced few peaks in the PMF. If our MS sample preparation workflow were alright, May I know what could be the possible causes?
P.S. Samples processed altogether was from a separate experiment; but their PMF signals were strong (and hence the high Protein Scores).
Thanks for your time, appreciated.
Kay
Hi Kay, hope all's well.
We have experienced this in the past ourselves and have attributed the lower molecular weight bands to fragments of the enzyme. This may be due to poor storage or lyophilisation of the enzyme product before it arrived in your lab. Additionally, review solubilisation of the enzyme in your lab to ensure all buffers are new. The commercial enzyme has most likely also autolysed and so I would encourage you to look at alternatives for the enzyme that have been chemically-modified so as to reduce autolysis - we now purchase a high quality trypsin that has been treated so as to reduce autolysis (it is dimethylated on lysine residues).
Finally, the "intense" bands that produced few peaks in the PMF may not ionise well (due to the properties of the residues present in those peptides) or may themselves degrade to smaller peptide sequences when analysed with a MALDI source. Do you have access to an ESI source or know someone who would be willing to run the same samples with a different instrument so that you can conclude fully what the bands are?
Good luck. Peter.
We have also encountered additional bands in commercials proteins. These could be products of proteolytic cleavage of protein either because of its intrinsic property. If good polyclonal antibody is available, Western blotting would give some information. All the best
Yes, could very well be fragments of the enzyme, see if anything in the literature regarding that for your enzyme. If you also pick up the bands with another antibody to your enzyme then good chance they are fragments of it (and your protein analysis supports that). That is what I work on, cleavage fragments and how their function is altered compared to the parent protein.
I think it is due to proteolysis and the fragments originate from the same enzyme due to storage or handling you could try to buy it from other commercial source or change the storage and shipping procedures with best luck
Although it seems that proteases could be culprit responsible for cutting the enzyme and giving additional band on the SDS PAGE but one of the reasons for the extra band of the same protein on SDS PAGE is due to protein folding. If the protein in Not properly folded and some molecules are in mis folded form they can appear as separate bands. That can be very clearly Identified on Non reductive PAGE.
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