Hi Antonio,

to search for +2 and +3 charged molecules is not simple, you have to deconvolute the spectrum first - for my knowledge, Agilent ofers a deconvolution software to create the "perfect" [M+H]+ spectrum, but other companies may offer such sofware, too. Second possibility: manual deconvolution of the most intesive signals, but this may consume tons of time... just search for Isotopic patterns with differences of 0.5 m/z or 0.33 m/z between the C12/C13 isotopes (if you have a signal of e.g. 1000, 1000.33, 1000.66 etc... this corresponds to a [M+3H]3+ ions with zero, one, or more C13 containing isotopes. The "deconvoluted" signal would be 2998, 2999 and 3000..., respectively. Calculate: [M+3H]3+ = 1000 m/z, [M+3H]+ = 3000 m/z, [M+H]+ = 2998 m/z. The molecule has the monoisotopic mass of 2997 in this case).

Hi, 

You wrote that you do not have an LC-MS system. Did you perform a MALDI analysis then?

Did someone perform the analysis for you?

As Marcus wrote, you need to deconvolute the spectra. Basically, you ran a scanning program which identify isotopic patterns and determine the charge for a each peak. 

I Among free tools that do this, you might want to try:

NISTS's AMDIS

http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:amdis

mMass 

http://www.mmass.org/

And finally the tools from the PNNL

http://omics.pnl.gov/software/decontools-decon2ls

I would bet on this last link, if I had to. It will probably allow you to control the output format, so that you have something ready to submit for a search.

Remember that the online MASCOT server can analyse only 100 queries at time.

Once you 

I forgot to add one thing.

X!Tandem: open search engine. You might want to try it.

ok, thank you, unfortunately I have no access to a MALDI, and my ESI is not coupled with an HPLC

... then the analysis will be much more complicated because you inject "everything" at a time into the ESI. HPLC could separate at least some peaks for you. Maybe try a 2D gel electrophoresis beforehand to have a better separation?

I don't think a 2D gel will have a dramatic impact.

An LC would really be the best solution in this case.

You might want to try a SCX fractionation, but you will still have peaks coming all together.

The last resource would be the following:

- load the tryptic peptide on a column trap (they are much cheaper than a whole column) at 95:5 H2O:ACN +0.1% formic acid.

Then elute with a 50:50 H2O:ACN +0.1% formic acid in a first run and with 5:95 H2O:ACN +0.1% formic acid. in a second run.

This should give a very basic separation, maybe enough to get some peptide separated.

HHi there

Mascot Wizard (free)

Mascot distiller

Both use raw data to deconvolute +2 +3 charged ions