PMF IDENTIFICATION MS/MS VALIDATION

MS/MS VALIDATION

-HN-CH-CO-NH-CH-CO-NH-R

_i

CH-R’

a

b

c

x

y

z

y

b

R”

d

v

w

low energy fragmentation high energy fragmentation

-HN-CH-CO-NH-CH-CO-NH-R

_i

CH-R’

a

x

b

y

c

z

y

b

R”

-HN-CH-CO-NH-CH-CO-NH-R

_i

CH-R’

a

x

b

y

c

z

y

b

R”

b

-a

= CO (28)

c

-b

= NH (15)

y

-x

= CO (28)

z

-y

= NH (15)

Informazioni complementari

y

-y

=NHCH(CHR’R’’)CO

b

-b

= NHCH(CHR’R’’)CO

The correct assignment of such a spectrum to a peptide

Sequence a first and central step in proteomic data processing.

MS/MS validation

A large number of computational approaches and software tools have been developed to automatically assign peptide sequences to fragment ion 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.

“Sequenziamento manuale” la sequenza aminoacidica viene dedotta direttamente dagli spettri MS o MS/MS.

“Sequenziamento manuale ???

Ma siamo matti? E allora a

che serve il computer?!?

Individuare gli ioni di sequenza della β-endorfina (MW=1860.14)

159.0922 (+) 186.07932 W Trp Tryptophan Tyr Y 163.06333 136.0762 (++) Tyrosine 129.114 (-) 156.10112 R Arg Arginine Phe F 147.06842 120.0813 (++) Phenylalanine 110.0718 (++) 137.05891 H His Histidine Met M 131.04049 104.0534 (+) Methionine Glu E 129.0426 102.0555 (+) Glutamic acid 101.1079 (84.08136) 128.09497 K Lys Lesine 101.0715 (+) 128.05858 Q Gln Glutamine Asp D 115.02695 88.03986 (+) Aspartic acid 87.05584 (+) 114.04293 N Asn Asparagine Leu L 113.08407 86.09698 (++) Leucine Ile I 113.08407 86.09698 (++) Isoleucine Cys C 103.00919 76.0221 (-) Cysteine Thr T 101.04768 74.06059 (+) Threonine Val V 99.06842 72.08133 (++) Valine Pro P 97.05277 70.06568 (++) Proline 60.04494 (+) 87.03203 S Ser Serine Ala A 71.03712 44.05003 (-) Alanine Gly G 57.02147 30.03438 (-) Glicine Immonium ion mass Residue mass Amino acids

[M+H] =1860.14 ∆m=1840.91-1727.83=113.08 86.09698 (++) 113.08407 L Leu Leucine fragment: b₁₇

[M+H]+_{=1860.14 ∆m=1727.83-1626.78 =101.05} fragment: b₁₆ 74.06059 (+) 101.04768 T Thr Threonine

[M+H] =1860.14 ∆m = 1626.78 – 1527.71 =99.07 fragment: b₁₅ 72.08133 (++) 99.06842 V Val Valine

[M+H]+_{=1860.14 ∆m = 1527.71 – 1317.58 = 210.13} fragment: b₁₃ 86.09698 (++) 113.08407 L Leu Leucine 70.06568 (++) 97.05277 P Pro Proline

[M+H] =1860.14 ∆m = 1317.58 – 1216.53 = 101.05 fragment: b₁₂ 74.06059 (+) 101.04768 T Thr Threonine

[M+H]+_{=1860.14 ∆m = 1216.53- 1088.47 = 128.06} fragment: b₁₁ 101.0715 (+) 128.05858 Q Gln Glutamine

[M+H] =1860.14 ∆m = 1088.47-1001.44 = 87.03 fragment: b₁₀ 60.04494 (+) 87.03203 S Ser Serine

Individuare gli ioni di sequenza del peptide RPKPQQFFGLM

MS/MS validation

129.114 (-) 156.10112 R Arg Arginine Phe F 147.06842 120.0813 (++) Phenylalanine Met M 131.04049 104.0534 (+) Methionine 101.1079 (84.08136) 128.09497 K Lys Lesine Gln Q 128.05858 101.0715 (+) Glutamine Leu L 113.08407 86.09698 (++) Leucine Pro P 97.05277 70.06568 (++) Proline Gly G 57.02147 30.03438 (-) Glicine Immonium ion mass Residue mass Amino acids b₁₀ b₉ b₈ b₈ b₇ b₆ b₅ b₄ b₃ b₂ b₁ 1329.65 1198.61 1085.53 1028.51 881.45 734.39 606.34 478.29 381.24 253.15 156.10 Y₁ Y₂ Y₃ Y₄ Y₅ Y₆ Y₇ Y₈ Y₉ Y₁₀ Y₁₁

M

L

G

F

F

Q

Q

P

K

P

R

b₁₁ b₁₀ b₉ b₈ b₇ b₆ b₅ b₄ b₃ b₂ b₁ 1329.65 1198.61 1085.53 1028.51 881.45 734.39 606.34 478.29 381.24 253.15 156.10 Y₁ Y₂ Y₃ Y₄ Y₅ Y₆ Y₇ Y₈ Y₉ Y₁₀ Y₁₁

M

L

G

F

F

Q

Q

P

K

P

R

SAMPLE

Remove protein from gel spot Enzymatically digest

all protein from spot MALDI of peptides mixture Search peptide mass 2D PAGE MALDI MS/MS

Protein MS/MS Ion Fragmentation pattern

MS/MS DATABASE SEARCHING

PROTEOMIC 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

TOP-DOWN

Protein Mixture

Excise spot; wash; digest

Run gel; stain; scan

Extract peptides Database search 2D SDS PAGE Simple peptides mixtures mass analyze

BOTTOM-UP

Advanced Sample

Preparation

… for Proteomics approaches

… for MALDI

Sample Dilution/Concentration

Dilute samples to the concentrations shown in the table below.

If the sample concentration is unknown a dilution series may

be needed to produce a good spot on the MALDI plate.

Note: highly dilute samples can be concentrated by Speed-Vacuum or Solid Phase Extraction.

Compound

_{Concentration}

0.1 to 10 pmol/µL

10 to 100 pmol/µL

Peptides and

proteins

Oligonucleotides

Polymers

100 pmol/µL

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

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

Typical contaminants in protein/peptide samples

No interference:

TFA, formic acid, β-mercaptoethanol, DTT, volatile

organic solvents, HCl, NH

OH, acetic acid

Tolerable: (< 50 mM)

HEPES, MOPS, Tris, NH

OAc, octyl glucoside

Avoid:

glycerol, sodium azide, DMSO,

SDS

, phosphate, NaCl,

2M urea, 2M guanidine

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

Drop Dialysis

•Fill a 250-400 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.

Use Millipore membrane, type VS, pore size 0.025 µM, diam. 25 mm

After

Before

Drop dialysis cleanup of Enolase

Yeast Enolase

(47 kDa) in 8 M

urea was

dialyzed for 1 hr

on a Millipore

membrane.

Sample Cleanup by Solid Phase

Extraction

• ZipTip - miniature C

column

chromatography

1. Sample Concentration and Buffer Removal

2. Fractionation

Assorbimento 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 una

fase inversa (tipo C18) sulla quale, dopo che i peptidi eluiti dall’in.gel digestion (sciolti in ambiente acquoso), gli stessi peptidi si legano. E’ possibile, con i peptidi legati alla resina, effettuare una serie di lavaggi in modo da eliminare eventuali contaminanti ed eluire successivamente i peptidi. (Si adottano le stesse strategie di eluizioni che si adottano in cromatografia HPLC a fase inversa. Questa procedura viene utilizzata soprattutto quando si hanno piccoli volumi e si vuole eliminare la presenza di sali.

•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

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.

Use of the C

₁₈

ZipTip

1. Sample Concentration and Buffer Removal

2. Fractionation

As peptides and proteins have differing affinities for C₁₈, the C₁₈ 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.

Step elution with Increasing ACN of

IgG HC Endo Lys C Digest from C

₁₈

tip

0 50000 100000 150000 C ounts 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 Mass (m/z) 10% ACN 30% ACN 50% ACN

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%.

In-Gel Digest

Fundamentals

• Handling the gel and slices

• Washing and destaining

• Enzymatic Digestion

• Peptide Extraction

• Concentration/Cleanup

• MALDI-TOF Analysis

In-Gel Digest Method

Success depends upon:

• Avoiding contamination of samples

• Digesting the protein efficiently

• Maximizing recovery of peptides

• Minimizing losses from handling

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.

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 dH₂0 (400 ul,

15 min.) Use gel loading tips to prevent accidental aspiration of gel pieces. This step washes away the soluble silver.

In-Gel Digest Method

Washing Destained Silver and Coomassie Gels

Trim the gel slices as needed to approx 1 mm3_{. 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 NH₄ 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 20-30 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).

In-Gel Digest Method

Enzymatic Digestion – Trypsin

Promega Sequencing Grade Modified Trypsin 10-15 ug/ml in 25 mM NH₄ Bicarbonate pH 8.0. Store at -70°C in one-time-use aliquots. (100 ul each)

Rehydrate the dried gels with approx. 10-15 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 16-24 hours. Convection oven is preferable to heat block.

In-Gel Digest Method

Extraction of Peptides

Soak the gel slice in 25-50 ul 50% ACN / 5% TFA for 30-60 min. with gentle agitation. Do not vortex.

Transfer the supernatant to a second clean tube .

Extract the gel again with another 25-50 ul aliquot of 50% ACN/5% TFA for 30-60 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 1.0-5.0 % TFA alone if ACN is undesirable, e.g. if ZipTip

In-Gel Digest Method

Reconstitution

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