If your mass analyzer is designed/tuned for the mass region (e.g., the quadrupole or trap is designed for the 0-5,000 Da region, then this is a very simple procedure. If your mass analyzer is designed for high MW or low MW work, then it may require sending out to a core facility that has the appropriately-designed instrument. Generally dissolve the peptide in CHCA matrix (50:50:0.1 acetonitrile/water/TFA) with 0.5 g/mL CHCA (for positive ion mode). Tune the collision cell to capture the mass of interest in the MS1 and then increase the fragmentation energy in the collision cell until you get the distribution of fragments you want. Typically, you get more information by summing the scans from a range of collision energies. If the analyzer is designed with a mass range that covers that mass, you might have to increase the duty cycle in the range of the mass of interest to get enough material for fragmentation.
Thank you Prof. Luke V Schneider and Prof. Bojidarka B. Ivanova
for your insightful comments.
Dear Rituparna,
Your instrument should be calibrated and optimized for the range eg;, 2-5 daltons before the analysis. Are you using the laser induced fragmentation to generate MS/MS? One important point to bear in mind is the isolation window for the MS1; if you have the window too wide, which in turn provides best sensitivity for allowing MS1 ions to be fragmented, be careful not to introduce peptide ions close to 4,300. Obtain a full scan and make sure that within the isolation window of your peptide of interest at 4.300 daltons, you do not have interfering peptides. If you do not, depending on the MALDI-TOF/TOF instrument, you can allow the maximum window (eg., plus and minus 5 mass units) on either side of 4,300 to achieve sensitivity before fragmenting the peptide. If you have interfering peptides on either side of 4,300, you have to take care to use a small window first eg., plus and minus 2 mass units (again, this depends on your instrument settings for MS/MS) and accumulate / add many MS/MS to get a good fragmentation spectrum.
Good luck,
Hediye.
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