Wee fun 1.2. Jonathan Katz

Transcription

1 Wee fun 1.2 Jonathan Katz

2 TMT1 Heard lots from Darren Here are a couple of brief points I show this data for 2 reasons: Convince you that 334 is our M Convince you that our product is C 8 H 14 O 5

3 CI DATA CI with CH3 -> M+H (1), M+C 2 H 5 (29), M+C 3 H 5 (41) 335, 363, 375 are all peaks don t see without CID

4 What do things weigh? What does Nitrogen weigh? I will wait as you check the periodic table Is it 14? ? ? Do CO and N 2 weigh the same? Both are 28. CO= N 2 = ? No! There are discrete compounds with discrete weights Just remember, 12 1 H s weigh more than 1 12 C and 16 1 H s weigh less than 1 16 O.

5 So With perfect accurate mass, you can identify the elemental composition of most anything! Sadly, not an easy task

6 Exact Mass of TMT1 substrate Sample a little too dirty for ICR or the two Magnetic sector machines But, this TOF Should be good enough. Calibrate right Before run and optimize for higher end

7 What does get us? We are expecting C 8 H 14 O 5 2H + 2Si(CH 3 ) 3 - CH 3 Or C 13 H 27 O 5 Si 2 =

8 Our product is the 1 st to be sane.. 1:4xSi? Would bring a load of 12 C s alone.. 2:seems unlikely because we can chemically methylate 2x which suggests at least 2 carboxyls. Of course it would be nice to have better mass measurement. But, the Bigger your compound the less error you have to have in order to do An elemental assignment If we could do a native mass we could do it Non-modified and it would be closer to 190

9 Why we do our own mass spec? Because we care.

10 Wading into the ocean: prmt3 ADMA methylator. Association with a tumor suppressor. X+prmt3 -> new prmt3 substrates (submitted) Multiple protein preps experiments need to be done. Prep s behave differently. The plasmid is showing an odd digestion pattern. Are things what we think they are?

11 How will this work if submitted

12 Calibrate.

13 10.99 prep

14 02.00 prep

15 This where we leave it? Rule one Provide the best sample possible Rule two Provide a better sample Can we work with this?

16 Zoom/Compare

17 See All we had to do was look close enough and do enough scans to guarantee that we would have a signal significantly above background Does this confirm the identity of the protein?

18 NO! Prmt3 should be ~ GST should be ~ = 68823? Or more like.. ~85000?

19 Buy it? Blow up 65000/85000

20 Didn t notice this til GM Remember that GM where I said visualization was everything?

21 Either way Would be nice to get a better full length sample So (remember, this is where caring matters!), I learned and used a few tricks and saw a new bag o fun

22 tricks Full scale and a blow up I buy the peak much more now..

23 So does this prove it? Random facts:.1% error on a MALDI-TOF not using a reflectron (>~8000).01% error on an electrospray Q3 but need it pure and A LOT MORE! So, how many proteins are ? At least 7! So how confirm?

24 Trypsin Digest. DTT treat, Iodoacetamide treat (reduce s-s bonds and keep them non-reactive) Dialize Trypsin Desalt Spot & go.

25 Trypsin Result prmt3 Calibrate WELL(!) Deisotope Use mass peaks to search

26 Cool What are the rest? Look at the sizes. How close can masses be? Search round 1.

27 Remove GST matches-research

28 Details Now I buy it.

29 Some thoughts Lots of spectra I didn t show you mostly making certain things work. Most importantly in my mind is the way to do calibrations Good protein identification (IMHO) seem most dependent on very low mass error. Many search programs (including the standard don t work as well as they could) None of this would have been obtained from just doing a sample submission.

30 Let s do it again: HSL7 Hsl7 is a negative regulator of Swe1 by promoting its degradation. Swe1 phosphorylates a cdc28/cyclin complex which halts the cell cycle. Hsl7 is homologous to JBP1 (a human SDMA methylator)

31 Hsl7 (cont d) To add confusion, Hsl7 always seems to copurify with 4 other bands. Is our activity due to Hsl7 or one of these others? Be nice to know what they are.

32 Predigest

33 Postdigest.

34 Band 2 Wide error.

35 Band 2 Narrow Error

36 But now out of range

37 Wanna confirm..? Ok..

38 Why these cleavages? COILS is a program that compares a sequence to a database of known parallel two-stranded coiled-coils and derives a similarity score. By comparing this score to the distribution of scores in globular and coiled-coil proteins, the program then calculates the probability that the sequence will adopt a coiled-coil conformation.

39 Thoughts The reason the 4 bands always purify with Hsl7 is because they are a part of Hsl7 In fact, it looks like the active form of Hsl7 might be a cleavage product Tina has been doing lots o partial digests and/or reactions with protease inhibitors that seem to further this theroy We will probably hear more in a couple weeks

40 Now what I am getting tired of reworking my data for powerpoint. So the question I ask myself what else do I want to talk about The rest of the stories are pretty incomplete from my end. So, we will continue with the protein ID theme and if I am motivated we will see some small molecule stuff

41 EF1alpha Involved in protein synthesis Has some known modifications: 30: mmk 79: tmk 316:dmK 390:mmK C term meth. These are modification Tanya is interested in looking at and so she has been purifying ef1alpha to do these studies.

42 2 MS questions. EF1alpha (cont d) Does Tanya have EF1alpha (Is that one band or two?) Can we check for modifications

43 Ok interpret Top is derived from fxn 8, Bottom from 16

44 Now Guess what I did? Fxn 8 Fxn 16

45 Fxn 8

46 Fxn 16

47 Targets Probably can see modified 30, 79 but the 316 and 390 look like they Might be too small Possibly with imperfect digestion or alternate Enzyme choice.

48 What See? mmk (K) STTTGHLIYK (C) (K) STTTGHLIYKCGGIDK (R) tmk (R) GITIDIALWK (F) (R) GITIDIALWKFETPK (Y) dmk (K) NVSVKEIR (R)

49 But with lots o mods (K)YQVTVODAPGHR(D) 1-tmK (K)GSFKYAWVLDK(L) Novel modification or is this just experimental variation? Which is correct, how check?

50 Distributions of Differences We have made determinations on aspects of the sizes of: Atoms, Proteins, Protein Fragments. Can still do: Alternate protein fragments and amino acids.

51 ------y5 = YNLTVLDAPGHR b4 -- = 492 (this is true based on MSMS Data PSD clearly needs A PSD calibration.. Then good

52

53 Other crap CNBr CPY ladders