1. The peptide bond is the weakest point, so the protein will break into two pieces.... After collision with helium the peptide will break into characteristic fragments

2. Example_1: Fragmentation of SLYK b-ionsy-ions SLY K Expected fragments: Total 7 peaks m/z I SLYK LYK YK K Y L S From the fragment spectra the peptide sequence can be generated

3. b-ionsy-ions SLY K Expected fragments: Total 7 peaks m/z I SLYK SLY SL Y K B-ions can help to identify the N-terminus of a peptide

4. b-ionsy-ions SLY K Expected fragments: Total 7 peaks m/z I SLYK LYK YK K m/z I SLYK SLY SL A completet spectra can be complex

5. How to interprete MS-spectra? 1. Try to find the C-terminal amino acid. 2. Try to find the next peak of the y-ion series in the spectra 3. Read the spectra from the tail to the head Strategy #1 Strategy #2 1.Find the peptide peak or the highest peak MW of one series. 2.Find the next peak in the series. 3.When the sequence is done, try to figure out in which way to read it. Combined Strategy 1.Find the b2/a2 pair 2.Read the b-ion series as far as you can 3.Read the y-ion series. 4.Try to match them together

6. Example:

7. Peptide C-terminal amino acid Δ113 Δ129 Δ101 Δ87 Δ233Δ128 Δ163

8. m/z A=71, P=97, V=99, I/L=113, N=114, D=115, M=131, H=137, F=147, K=129/147, Q=128, S=87 Y=163, E= 129 T=101 R=175 phosphorylation = +80 ICPL= +86/92 List of masses of amino acid and modifications

9. R I/L E T S K(12C) K (13C) Q Y 215 173 Sequence: YQKSTE(I/L)(I/L)(I/L)R ICPL-

10. A, Overexpression and enhanced activity of the epidermal growth factor receptor (EGFR) is often observed in human cancers. EGFR is a tyrosine phosphotransferase (kinase) and abnormal EGFR-kinase activity is related to poor prognosis. Upon binding of the ligand EGFR undergoes autophosphorylation (self phosphorylation), an important step in EGF signal transduction. Figure 1 shows the fragment spectra of the peptide (PDYQQDFFPK) generated from isolated EGFR. Please confirm the predicted sequence and mark the phosphorylation site. Figure 1: Fragment spectrum. (m/z A=71, P=97, V=99, I,=113, N=114, D=115, M=131, H=137, F=147, K=129/147, Q=128, Y=163, phosphorylation = +80) B, A pharmaceutical company wants to produce inhibitors against EGFR-kinase for cancer therapy. Please explain briefly how mass spectrometry can help to identify an inhibitor compound.