1. 100526 Training From MS scan to SIM

2. Work Flow MS Scan Use Qual anal to determine which parent ion to look at  use the most abundant ion, usually the 2+ or the 3+ SIM for determining the “fragmentor”

3. 3. When purging, open the MS scan method that we’ll be using. Double check the solvent. 2. Purge lines, nano and Cap, right click and hit “purge” Green: standby, pink: waiting, blue: in use. When purging, the QQQ should be standby. The instrument will auto turned on when done purging. 1. Load the chip, hit “operate”

4. Always make sure before start, your chip is default at enrichment Note that it needs a time point.

5. 4. Monitoring the total ion here! 5. Make a new folder under D drive>masshunter>data. Open a new worklist, add blank, put in blank sample to autosampler, run method MS scan 001.

6. 6. Run> run worklist, columns should turn blue~~

7. Save method! Refers to 100526 note

8. From MS Scan to SIM The sample for training contains 9 diff. peptides, ea. of them has the phosphorylated form and non-phosphorylated form (the sample is phosphatase treated!). We’ll be looking at the cdh1pS16 and Fin1pS148 Use ‘proteinprospector’ to determine the m/z of duplet/triplet/+4/etc. charged ions.

9. Max charge = 4 EAIyAAPFA  phosphorylated Tyr EAIY[50]AAPFA  add in special peptide mass

10. Most likely! Av, isotopic ave m/z, mono, the most abundant one

11. d 1. Extract MS spectra 2. Or look for the ions Do delete the BPC (base peak chromatogragh) 3. Write down 1.the observed m/z (661.6 & 991.7) 2.The corresponding charge (+2 & +3) 3.Elusion time 7.3-7.6 On printed out proteinprospector sheet This time we will be using 661.6 for Fin1. 4. 806.6 for cdh1 (+2)

12. 806.6 for cdh1 (+2)

13. Open method>SIM80~SIM180, we do 80-120 this time Change the parameters from this pre-saved methods We can run up to 4 diff compounds at a time, but ea. Run, the fragmentor can not be changed. Internal standard peptide? Changed automatically, depending on how many compounds you put to run~ MS2 SIM!!!

14. Open data files, look at the peak area and pick the best frag. volt. This time, 120 is the best Peak area: 54038

15. From SIM to PI_Scan _CE Modify and save the PIScanCE methods Usually goes from 15  30, super small peptide starts at 10, the thorough range would be 5- 35. CE=Collision energy

16. Add a script because we won’t be here when the run gets to the end. Always run two blanks after HSA standard. Because that thing is stickyyy! Precursor ion Record the pressures on the log book