I have some questions regarding how to read a MALDI mass spectrum in a paper. I am a biologist by training, so I would like some analytical chemists to help me out.
The researchers ran a western blot, and immunoblotted for a protein of interest. They cut out this band from the western blot, subjected the protein to trypsin digest, and used these peptide fragments for MALDI-TOF analysis. I know for a fact there will be other proteins at the same apparent molecular mass which would have also been included in the analysis, which should be taken as contaminants in the resulting spectrum.
They produced a graph with the absorbance intensity x 1,000 on the dependent axis, and mass:charge (m/z) ratio on the independent axis.
The authors point to two peaks, which they say correspond to two peptide fragments from the tryptic digest of their protein of interest. They provide the supposed peptide fragment sequences and m/z ratio data for these two peaks, and I checked to make sure they are the same as reported. The authors expect to see two peaks: protein of interest is digested to two fragments, A + B.
I have two questions:
Peak 'A' and peak 'B' are of different intensities. Peak 'A' is 20-fold greater than peak 'B'. However, the tryptic digest must yield equimolar amounts of peptide fragments A and B. Is it possible that these intensity peaks can be 20-fold different? Wouldn't the absorbance intensity correlate to total analyzed peptide mass?
Is it normal to multiple absorbance intensity by a (seemingly arbitrary) factor, in this case, 1000? Since the absorbance intensity of peak 'B' is < 0.5 (after scaling up), could this represent noise rather than signal in the analysis?