PMF IDENTIFICATION MS/MS VALIDATION

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1 PMF IDENTIFICATION MS/MS VALIDATION

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5 rammentazione dei Peptidi x n-i v n-i y n-i z n-i y n-i-1 -HN-CH-CO-NH-CH-CO-NH- R i a i b i c i wn-i CH-R R i+1 i+1 b i+1 low energy fragmentation d i+1 high energy fragmentation

6 rammentazione dei Peptidi x n-i y n-i z n-i y n-i-1 -HN-CH-CO-NH-CH-CO-NH- R i a i b i c i CH-R R i+1 i+1 b i+1

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8 rammentazione dei Peptidi Informazioni complementari x n-i b 1 -a 1 = CO (28) c 1 -b 1 = NH (15) y 1 -x 1 = CO (28) z 1 -y 1 = NH (15) y 2 -y 1 =NHCH(CHR R )CO b 2 -b 1 = NHCH(CHR R )CO -HN-CH-CO-NH-CH-CO-NH- a i R i b i c i y n-i y z n-i-1 n-i CH-R R i+1 i+1 b i+1

9 S/MS validation he large correct number assignment of computational of such a approaches spectrum to and a peptide software tools have equence en developed a first to and automatically central step assign in proteomic peptide sequences data processing. to fragment n spectra. These can be classified in three categories: Database searching, where peptide sequences are identified by correlating acquired fragment ion spectra with theoretical spectra predicted for each peptide contained in a protein sequences database. De novo sequencing, where peptide sequences are explicitly read out directly from fragment ion spectra. Hybrid approaches, such as those based on the extraction of short sequence tags of 3-5 residues in length, followed by error tolerant database searching.

10 Sequenziamento manuale la sequenza aminoacidica viene dedotta irettamente dagli spettri MS o MS/MS. Sequenziamento manuale??? Ma siamo matti? E allora a che serve il computer?!?

11 MS/MS Frammentazione dei Peptidi Individuare gli ioni di sequenza della β-endorfina (MW= )

12 Amino acids Glicine Alanine Serine Proline Valine Threonine Cysteine Isoleucine Leucine Asparagine Aspartic acid Glutamine Lesine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan Gly Ala Ser Pro Val Thr Cys Ile Leu Asn Asp Gln Lys Glu Met His Phe Arg Tyr Trp G A S P V T C I L N D Q K E M H F R Y W Residue mass Immonium ion mass (-) (-) (+) (++) (++) (+) (-) (++) (++) (+) (+) (+) ( ) (+) (+) (++) (++) (-) (++) (+)

13 [M+H] + = m= = Leucine Leu L (++) fragment: b 17

14 [M+H] + = m= = Threonine Thr T (+) fragment: b 16

15 [M+H] + = m = =99.07 Valine Val V (++) fragment: b 15

16 [M+H] + = m = = Leucine Leu L (++) Proline Pro P (++) fragment: b 13

17 [M+H] + = m = = Threonine Thr T (+) fragment: b 12

18 [M+H] + = m = = Glutamine Gln Q (+) fragment: b 11

19 [M+H] + = m = = Serine Ser S (+) fragment: b 10

20 S/MS validation ndividuare gli ioni di sequenza del peptide RPKPQQFFGLM Spettro teorico

21 Amino acids Glicine Proline Leucine Glutamine Lesine Methionine Phenylalanine Arginine Gly Pro Leu Gln Lys Met Phe Arg G P L Q K M F R Residue mass Immonium ion mass (-) (++) (++) (+) ( ) (+) (++) (-) Y 11 Y 10 Y 9 Y 8 Y 7 Y 6 Y 5 Y 4 Y 3 Y 2 Y R P K P Q Q F F G L M b 1 b 2 b 3 b 4 b 5 b 6 b 7 b 8 b 8 b 9 b 10

22 Y 11 Y 10 Y 9 Y 8 Y 7 Y 6 Y 5 Y 4 Y 3 Y 2 Y P K P Q Q F F G L M b 1 b 2 b 3 b 4 b 5 b 6 b 7 b 8 b 9 b 10 b 11

23 OP-DOWN APPROACH: MS/MS validation SAMPLE Remove protein from gel spot 2D PAGE MALDI of peptides mixture Enzymatically digest all protein from spot Search peptide mass MALDI MS/MS Protein MS/MS Ion Fragmentation pattern MS/MS DATABASE SEARCHING

24 ROTEOMIC APPROACHES 1. Top down approach 2. Bottom up or shotgun approach Top down approach: a protein is separated from a complex mixture, purified and identified by direct fragmentation by mass spectrometry Bottom up approach: a mixture of protein is first made more complex through enzymatic digestion, e.g. trypsin, followed by liquid chromatography tandem MS to identify the peptide fragments.

25 OP-DOWN Protein Mixture Run gel; stain; scan Excise spot; wash; digest Simple peptides mixtures 2D SDS PAGE xtract peptides mass analyze Database search

26 OTTOM-UP Protein Mixture Proteolysis Multidimensional LC MS and MS/MS Complex peptides mixture MS/MS Database search

27 dvanced Sample reparation for Proteomics approaches for MALDI

28 Sample Dilution/Concentration ilute samples to the concentrations shown in the table below f the sample concentration is unknown a dilution series may e needed to produce a good spot on the MALDI plate. Compound Peptides and proteins Concentration 0.1 to 10 pmol/µl Oligonucleotides Polymers 10 to 100 pmol/µl 100 pmol/µl Note: highly dilute samples can be concentrated by Speed-Vacuum or Solid Phase Extraction.

29 Sample clean-up Removal of buffer salts, urea, guanidine, EDTA, glycerol, DMSO, detergents, etc. Dilution Washing Drop dialysis Cation exchange Pipette tip column chromatography ZipTips

30 Sample Dilution Simplest way to minimize effect by contaminants. Goal is to dilute contaminants to the point where they no longer interfere with analysis of sample. Requires high enough analyte concentration in sample to provide acceptable data when diluted out.

31 Typical contaminants in protein/peptide samples No interference: TFA, formic acid, β-mercaptoethanol, DTT, volatile organic solvents, HCl, NH 4 OH, acetic acid Tolerable: (< 50 mm) HEPES, MOPS, Tris, NH 4 OAc, octyl glucoside Avoid: glycerol, sodium azide, DMSO, SDS, phosphate, NaCl, 2M urea, 2M guanidine

32 On-Plate Washing Buffer and Salt Removal Dry sample and matrix Deposit 1-2 ml cold 0.1% TFA Leave on for 5-10 sec., then remove Detergent contamination Use 5% Isopropanol Cell Extract Contamination Use 100% Isopropanol

33 Drop Dialysis To remove low molecular weight contaminants Use Millipore membrane, type VS, pore size µm, diam. 25 mm Fill a ml container with deionized water. Float the membrane on the water (shiny side up). Place about 10 ml of sample solution on the membrane. Add 1mL ACN to the sample spot to increase surface area. Allow to sit for ~45 minutes. Remove an aliquot with pipette, add matrix and spot plate.

34 Drop dialysis cleanup of Enolase Before Yeast Enolase (47 kda) in 8 M urea was dialyzed for 1 hr on a Millipore membrane. After

35 Sample Cleanup by Solid Phase Extraction ZipTip -miniature C 18 column chromatography 1. Sample Concentration and Buffer Removal 2. Fractionation 3. Affinity Experiments

36 ssorbimento su Reversed Phase (C18) di peptidi generati da in gel digestion ZipTip: puntali per pipette eppendorf alla cui punta è impaccata una resina che supporta un fase inversa (tipo C18) sulla quale, dopo che i peptidi eluiti dall in.gel digestion (sciolti i ambiente acquoso), gli stessi peptidi si legano. E possibile, con i peptidi legati alla resin effettuare una serie di lavaggi in modo da eliminare eventuali contaminanti ed eluir successivamente i peptidi. (Si adottano le stesse strategie di eluizioni che si adottano i cromatografia HPLC a fase inversa. Questa procedura viene utilizzata soprattutto quando hanno piccoli volumi e si vuole eliminare la presenza di sali.

37 Procedure for using C 18 ZipTips Condition the ZipTip with 10 µl of acetonitrile (ACN), then 10 µl of 50% ACN/0.1% TFA, then 2 x 10 µl of 0.1% TFA. Load the sample onto the ZipTip by pipetting 5-10 µl sample up and down several times and discarding the liquid. Wash C 18 tip with 3 x 10 µl of 0.1% TFA to remove salts. Elute the sample from the ZipTip with 30-70% ACN or elute directly into the matrix (e.g. CHCA in 50% ACN/0.1%TFA); minimal volume of ~3 µl can be used.

38 Use of the C 18 ZipTip 1. Sample Concentration and Buffer Removal 2. Fractionation As peptides and proteins have differing affinities for C 18, the C 18 tips can be used to fractionate mixtures according to their hydrophobicities. Increasing the ACN in a step gradient of 10% - 50% in the eluent increased the number of peptides seen. By fractionating a peptide mass map this can also be beneficial for PSD analysis. 3. Affinity Experiments

39 Step elution with Increasing ACN of IgG HC Endo Lys C Digest from C 18 tip % ACN Counts % ACN % ACN Mass (m/z) Most peptides are seen at 30 % which is a good concentration to use for most digests as this can be used to remove Coomassie Blue which elutes at 40%.

40 In-Gel Digest Fundamentals Handling the gel and slices Washing and destaining Enzymatic Digestion Peptide Extraction Concentration/Cleanup MALDI-TOF Analysis

41 In-Gel Digest Method Success depends upon: Avoiding contamination of samples Digesting the protein efficiently Maximizing recovery of peptides Minimizing losses from handling

42 In-Gel Digest Method Handling the Gel and Slices Gloves and lab coats must be worn at all times to avoid keratin contamination. Work on a clean surface. Use clean polypropylene microcentrifuge tubes, 500 or 1500 ul with snap caps. Test first to confirm OK (i.e., does not leach out polymers, mold release agents, plasticizers, etc.) Set aside a box for digest use only, handle only with gloves. Use only clean tools, containers and reagents for anything that will come in contact with the samples. Keep samples capped at all times unless being processed.

43 In-Gel Digest Method Silver Stained Gels Non-destructive Silver stained samples should be destained prior to analysis as follows: Prepare stock solutions of 30 mm Potassium Ferricyanide and 100 mm Sodium Thiosulfate. Store each at 4 C for up to 3 months. Make the working destain solution immediately prior to use by mixing the two stock solutions above at a 1:1 ratio. Soak gel slices in 100 ul destain solution for 10 minutes. This step converts the silver to a water soluble form. The gel will clear. Carefully remove the destain solution and wash 3X in dh 2 0 (400 ul, 15 min.) Use gel loading tips to prevent accidental aspiration of gel pieces. This step washes away the soluble silver. Ref: Electrophoresis 1999, 20,

44 In-Gel Digest Method Washing Destained Silver and Coomassie Gels Trim the gel slices as needed to approx 1 mm 3. Run a negative and positive control, as well as a reagent control (containing no gel slice). Transfer gels to 500 or 1500 ul capped microcentrifuge tubes Wash gels 3X in 50% ACN / 25 mm NH 4 Bicarbonate ph 8.0 (400 ul, 15 min. each time). This will remove excess Coomassie Blue. Soak in 100% ACN for 5 min. to dehydrate the gels, they will turn opaque white. Remove the ACN. (Note: Be sure that the ACN used does not contain any acid, otherwise the ph will be incorrect. Dry gels in Speed-Vac for min. This will shrink the gels. (Be sure that the inside of the Speed-Vac is clean and free of particulates. Do not allow anyone to use the Speed-Vac with ungloved hands during this step as sample tubes will be uncapped).

45 In-Gel Digest Method Enzymatic Digestion Trypsin Promega Sequencing Grade Modified Trypsin ug/ml in 25 mm NH 4 Bicarbonate ph 8.0. Store at -70 C in onetime-use aliquots. (100 ul each) Rehydrate the dried gels with approx ul cold Trypsin solution. The gels will swell and turn clear. Check after 30 min. for sufficient volume to completely wet entire gel. Add additional Trypsin if needed for large gel pieces. There is no need to overlay with additional buffer. Incubate tightly capped at 37 C for hours. Convection oven is preferable to heat block.

46 In-Gel Digest Method Extraction of Peptides Soak the gel slice in ul 50% ACN / 5% TFA for min. with gentle agitation. Do not vortex. Transfer the supernatant to a second clean tube. Extract the gel again with another ul aliquot of 50% ACN/5% TFA for min. Combine the two extracts and Speed-Vac to complete dryness, about 1 hour. Note: dry at room temp or heat to no more than 30 C. Drying can also be done in a lyophilizer. (Note: Peptides can alternatively be extracted from the gels with % TFA alone if ACN is undesirable, e.g. if ZipTip cleanup will be used.)

47 In-Gel Digest Method Reconstitution MALDI-TOF Analysis

48 Staining Procedure Results have shown that Coomassie Blue should be used if the sensitivity is adequate as the recovery of peptides is better than with Silver Staining. Excising the Gel Spot Care should be taken to cut precisely around the stained area to prevent any unnecessary contamination.

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