Maldi PTM Filter

Modification detection/quantification in samples with unknown modified and unmodified peptides

Workflow:

1. MS run on the MALDI-TOF/TOF

• Four modes: linear positive, linear negative, reflector positive, reflector negative (i.e. four sets of spectra per sample spot)

2. Change data format

• In MALDI software: Export to .t2d format

• Convert .t2d file to ASCII or process using a modification of an ABI provided macro called "CreateMWIntensityList." This calculates isotope cluster areas for all peaks and exports a text file. Our modifications make it sort according to mass instead of intensity and list the top 3000 peaks instead of the top 30. These modifications are crucial to making sure the filter program can find thiophosphopeptides.

3. Run through the filter program to pick thiophospho-modified peaks

• Use rules*

• Generate hit list table of m/z values (text/Excel file) including quantitation of relative peak areas for “modified” and “unmodified” (as % modified) for each hit, for each instrument mode spectrum

*see attached images for spot and filename info and scheme for rules

4. Copy refl pos peaklist into an MS/MS pos run for peptide identification

5. Copy refl neg peaklist into MS/MS negative run for validation of thiophosphorylation (gives a peak in the neg mode MS/MS spectrum at 95 amu)

*maldi spot format:

*rules for finding thiophosphorylation (simple):

• B1_LINEAR_2.txt: Linear negative test spectrum

• B1_MS_1.txt: Reflector positive test spectrum
  • Known Pb's (thiophosphopeptides) at 1771 and 1929
  • Known Pa's at 1677.8846 (IPGSTAAEAIYAAPFAK) and 1833.9857 (RIPGSTAAEAIYAAPFAK)

• ap10predicteddigest.pdf: Predicted peptides for this protein

* detail of fragmentation between modes:

* Larger zoomed back view, same sample both reflector positive (top) and linear negative (bottom) spectra: note fragments for three peaks:

* Detail of fragment peak in reflector positive: look at peak cluster shape, note overlapping parts--these are all related to the fragment ion:

* This is in contrast to this "normal" peptide peak, it does not overlap with that characteristic pattern like the fragment ion does:

• 4-17-elute-3-linneg.txt: test spectrum processed with altered ABI macro

• 4-17-elute-3-refpos.txt: test spectrum processed with altered ABI macro

• 4-17-elute-3-linnegASCII.txt: ASCII exported entire spectrum

• 4-17-elute-3-refposASCII.txt: ASCII exported entire spectrum