Hi, I have purified a protein by various chromatographic techniques and confirmed with SDS PAGE analysis. Further I gave that for MALDI sequencing. Hence, the sequencing result shows several peaks rather then expected. In addition MALDI performed without digestion. So, what will be the reason for several peaks. Spectrum attached for your reference
Hi, Shiyam -
Can you clarify your question a bit? For example, sending a sample for sequencing implies that the protein was digested, but later it's noted that the sample was 'without digestion'.
In any event, the attached spectrum mainly indicates that the sample was not very pure, which is not unusual, i.e., it's rare to have a completely pure protein.
Do you know anything about the 'expected' protein, e.g., does the main signal in the mas spectrum seem reasonable?
Thanks!
Hi Shiyam,
few things: 1) I see the data are a snapshot from flexAnalysis software, which tells me your facility uses one of the Bruker MALDI-TOF instruments. This is what I work on in my lab, and I am speaking based on 15 years of experience. Sequencing of such small protein (~6000Da) can be done without digestion by either FAST (Fragmentation Analysis and structural TOF), ISD (in-source decay), T3-sequencing or LIFT methods available on advanced Bruker MALDI-TOF models with TOF/TOF capabilities.
2) The attached spectrum, however, does not look like a fragmentation spectrum of any kind, which suggest that only MW measurement have been done on this sample. This in turn means that facility might have misunderstood the goal of analysis and thought that all you wanted was MW confirmation.
3) It is possible that your MS facility was not able to sequence your sample because they observed these many peaks in close proximity to the mass of interest (if they knew the mass), or could not choose which peak to sequence (if the did not know what mass to look for). Thus, you may want to work with them a bit closer on this.
4) Why multiple peaks? Few peaks on HPLC "purified" protein is very common as John noted above because protein may exist in a few isoforms that frequently co-migrate and co-elute during separation. Also possible is a partial degradation of sample during storage post separation (more common that you would think!), or artificial degradation of the protein in the gas phase during MALDI ionization. Likewise, posttranslationally modified proteins often showup in both forms - with and without PTMs and may co-elute in HPLC and then create a few peaks in the MS spectrum.
5) Prior info on the protein/sample will be very helpful to the MS facility staff who works with you on the MALDI analysis. If amino acid sequence is known, the MW can be calculated. If hints on possible PTMs are available, then mass shifts between detected peaks in your attached spectrum could be inspected on the subject of PTM fit. Likewise, neutral loss from the molecular ion is very common and some of those peaks may account for that, so ask the staff to look for that.
6) Not relevant to your case, but for your info in principle, multiple peaks in the spectrum of purified protein can be observed when measurement is done at high enough resolution to resolve monoisotopic masses arising from inclusion of naturally occurring isotopes like carbon 13, for example, into small portion of protein molecules. This is not the case with your spectrum, though, because peak pattern and mass differences between them do not correspond to typical monoisotopic cluster. I bet the spectrum you show is acquired in linear mode where one would see one average centroid peak per compound.
Hope this helps and good luck with your research.
Thanks Mr. John & Ms. Elena for your valuable suggestion.
Thanks once again
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