Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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Step 8: Spot Excision384

Tab. 8.1: K2C1 Human: keratin, type II cytoskeletal 1 (K1 skin).

Nominal mass (M

): 65847; calculated pI value: 8.16.

Position in

sequence

Experimental

mass [M+H]

Theoretical

mass [M]

Sequence

185–196 1382.68 1383.69 SLNNQFASFIDK

211–222 1474.75 1473.74 WELLQQVDTSTR

417–431 1716.85 1715.84 QISNLQQSISDAEQR

223–238 1993.98 1992.97 THNLEPYFESFINNLR

518–548 2383.95 2382.94 GGGGGGYGSGGSSYGSGGGS-

YGSGGGGGGGR

549–587 3312.31 3311.30 GSYGSGGSSYGSGGGSYGSGG-

GGGGHGSYGSGSSSGGYR

Tab. 8.2: K2E keratin (Dandruff), 67K type II epidermal – human.

Nominal mass (M

): 65825; calculated pI value: 8.07.

Position in

sequence

Experimental

mass [M+H]

Theoretical

mass [M]

Sequence

245–253 1037.50 1036.49 YLDGLTAER

381–390 1193.60 1192.59 YEELQVTVGR

46–61 1320.60 1319.59 HGGGGGGFGGGGFGSR

71–92 1838.90 1837.89 GGGFGGGSGFGGGSGFGGGSGFS-

GGGFGGGGFGGGR

93–128 2831.20 2830.19 SISISVAGGGGGFGAAGGFGGR

Tab. 8.3: K.9. keratin 9 (skin), type I, cytoskeletal – human. Nominal

mass (M

): 61950; calculated pI value: 5.14.

Position in

sequence

Experimental

mass [M+H]

Theoretical

mass [M]

Sequence

233–239 897.40 896.39 MTLDDFR

224–232 1060.60 1059.59 TLLDIDNTR

242–249 1066.50 1065.49 FEMEQNLR

449–471 2510.10 2509.09 0 EIETYHNLLEGGQEDFESSGAGK

63–94 2705.20 2704.19 GGGSFGYSYGGGSGGGF-

SASSLGGGFGGGSR

Step 8: Spot Excision 385

Tab. 8.4: K10 (dandruff) keratin 10, type I, cytoskeletal – human.

Nominal mass: 59492; calculated pI value: 5.17.

Position in

sequence

Experimental

mass [M+H]

Theoretical

mass [M]

Sequence

442–450 1165.60 1164.59 LENEIQTYR

323–333 1365.60 1364.59 SQYEQLAEQNR

166–177 1381.64 1380.63 ALEESNYELEGK

41–59 1707.80 1706.79 GSLGGGFSSGGFSGGSFSR

423–439 2024.94 2023.93 AETECQNTEYQQLLDIK

208–228 2366.26 2365.25 NQILNLTTDNANILLQIDNAR

Manual procedure Wearing powder-free gloves, use a clean scalpel

blade to cut around the protein spot of interest. Attempt to take as

little of the surrounding gel as possible. Using a surgical needle

transfer the excised gel piece to a pre-rinsed 500-mL Eppendorf tube.

387

Step 9: Sample Destaining

Generally three methods are used for gel staining of 2-D gels. Coo-

massie brilliant blue, silver and Sypro Ruby.

9.1

Coomassie Blue-stained Protein Spots

The CBB will heavily suppress signal in both MALDI and ESI analy-

sis. CBB will be easier to remove before digestion rather than after,

because after digestion the CBB will be concentrated along with the

peptide digest during the extraction stage. The CBB will be very diffi-

cult to remove by a microscale purification step (see Step 10).

Add 25 mL of 75 mmol/L ammonium bicarbonate (40% ethanol) to

the excised Coomassie stained gel plug. Vortex and leave to stand.

The supernatant will rapidly turn blue, remove supernatant after ten

minutes and replace with a further aliquot of the destain solution

until the excised spot is destained. The gel plug is now ready for

digestion.

9.2

Silver-stained Protein Spots

The silver staining method has to be compatible with MS analysis.

Sensitive silver stain methods, in the order of 1–10 ng per protein

spot (Rabilloud, 1999), typically required the use of glutardialdehyde

as part of the sensitization process. However, glutardialdehyde reacts

with the amino groups of proteins, both e-amino (lysine side chain)

and the a-amino, cross-linking the protein to the gel. This step needs

to be eliminated for optimal MS analysis (Shevchenko et al. 1996; Yan

et al. 2000; Sinha et al. 2001). Further optimization to the procedure

was reported by Gharahdaghi et al. (1999) by removing the silver ions

prior to digestion.

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

The length of time required to

destain will be dependant on

the intensity of the stain.

Step 9: Sample Destaining388

1. Prepare fresh solutions of potassium ferricy-

anide (30 mmol/L) and sodium thiosulfate

(100 mmol/L).

2. Prepare a 1:1 solution of the above reagents and

immerse the excised spots in this newly pre-

pared solution.

3. Once the dark stain has been removed, wash the

spot with water.

4. Equilibrate the excised spot in ammonium

bicarbonate (200 mmol/L) for 15 minutes,

remove supernatant and replace with a second

aliquot of ammonium bicarbonate and leave

for 15 minutes.

5.The gel plug is now ready for digestion.

Volume used depends on the

size of the excised gel plug.

389

Step 10: Protein Digestion

10.1

In-gel Digestion

Perform the reduction and alkylation step prior to digestion. Remem-

ber to use the same alkylation reagent as was used between the first

and second dimensions of 2-D electrophoresis.

1. Reduction. Add 10 mL of dithiothreitol solution

(5 mmol/L in 25 mmol/L ammonium bicarbo-

nate) to the gel plug (or sufficient to cover the

gel piece) and incubate for 30 minutes at 60 C.

2. Alkylation. Add 10 mL of iodoacetamide

(55 mmol/L in 25mmol/L ammonium bicarbo-

nate) to the gel plug and stand at room tempera-

ture for 30 minutes in the dark.

3. Remove the supernatant, wash with ammo-

nium bicarbonate (25 mmol/L), remove super-

natant and wash with acetonitrile.

4. Take the excised gel plug and cut into smaller

pieces with the scalpel (2–4 mm

). Press down

on the gel plug with the surgical needle holding

the gel plug in place; allowing the cutting of the

plug. Transfer the gel plug pieces to the rinsed

Eppendorf tube.

5. Dehydration step. Dehydrate gel plug with acet-

onitrile (325 mL, 10 minutes each). The gel

plug will begin to look white, after three washes

it will be completely white.

6. Dry plug further in a speed vac or drying cham-

ber, until gel plug appears “dust like.”

7. Rehydration step. Apply enzyme in buffer

(10 mLat40ng/mL in 50 mmol/L ammonium

bicarbonate) to the dried gel plug and incubate

on ice for 45 minutes.

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Step 10: Protein Digestion390

8. Digestion. After 45 minutes remove superna-

tant and add enough buffer solution (without

enzyme) to cover the hydrated gel piece.

9. Incubate at 30 C, for between 1 hour and over-

night (a preliminary peptide mass fingerprint

can be obtained after 1 hour by sampling from

the digestion mixture).

10. Extraction step. Add 20 mL of 50 mmol/L

ammonium bicarbonate and sonicate for

10 minutes. Add 20 mL of acetonitrile : 5% TFA

(1:1) and sonicate for 10 minutes. Remove

supernatant and dispense into a separate tube.

11. Add 20 mL of acetonitrile : 5% TFA (1:1) and

sonicate for 10 minutes. Combine supernatant

extracts. Repeat one further time.

12. Add 10 mL acetonitrile and sonicate. Remove

and combine with earlier supernatant extracts.

10.2

Non-gel Digestion

Two methods can be considered.

A. Split the sample in to two fractions, 4% CHAPS-soluble and 4%

CHAPS insoluble. The samples can then prepared according to the

following procedure:

1. Dissolve approximately 1 mg of protein in 1 mL

of 9 M urea with 50 mmol/L DTT, and then

incubate for 60 min at 20 C.

2. Add 1 mL of 8 M urea, 250 mM TrisHCl, pH

8.8, and 125 mM iodoacetamide, and then incu-

bate for 60 min at 20 C.

3. Buffer exchange 1 mL of the mixture with

20 mmol/L ammonium bicarbonate, pH 7.8, on

a NAP-10 desalting column.

4. Digest the desalted protein sample with trypsin

(concentration ratio of 50:1) for 2 h at 37 C.

5. Inactivate the trypsin by adding formic acid to

the sample.

10.2 Non-gel Digestion 391

B. Alternatively:

1. Denature sample by dissolve it in 0.5 mL

8 mol/L (GUA-HCl) 50 mM ammonium bicar-

bonate,

2. Reduce by adding 20 mL 0.8 M PlusOne DTT

(10 min at 37 C) and alkylate with 25 mL

0.5 mol/L iodoacetic acid (10 min at 37 C).

3. Exchange the buffer to 50 mM ammonium

bicarbonate on NAP-5 desalting columns and

digest using Ettan Trypsin sequencing grade, at

pH 8.4 for12 h at 37 C.

393

Step 11: Microscale Desalting and

Concentrating of Sample

The sample preparation for MALDI (and ESI) is a crucial step. The

analyte must be incorporated into the matrix crystals, a process that

is significantly upset by the presence of contaminating salts and buf-

fers (see table 1 for MALDI salt/buffer compatibility). Sample pre-

paration can be tailored to a particular matrix. For instance a-cyano-4-

hydroxycinnamic acid is insoluble in water. Sampling directly from

the digestion mixture, the analyte solution can be spotted onto a pre-

formed thin layer of matrix on the MALDI target (thin film method).

Once the dried spot has formed, the spot can undergo significant

washing with 0.1% TFA solution on the target surface, removing the

salt contamination. As the analyte has been incorporated into the

matrix crystal, it is preferably bound during the washing step (Vorm

et al. 1994). Addition of nitrocellulose to the matrix solution allows

for improved desalting and improved binding on the MALDI target

(Shevchenko et al. 1996).

In contrast, 2,5-dihydroxybenzoic acid is a water soluble matrix. It

is possible to sample directly from the digest mixture without any

sample clean up (on target washing is not applicable) as the matrix

excludes the contaminants from the crystallization process

However if sampling from the digest mixture is not applicable (or

if this approach is not attractive) then the peptides are extracted as

detailed in the section above. However, the combined extract volume

(~100 mL) may be too dilute for successful analysis of the peptide mix-

ture; in these instances the sample must be concentrated prior to ana-

lysis. Discussed earlier, simply concentrating the sample will concen-

trate all the contaminants as well, hence a clean up step is recom-

mended. Kussman et al. (1997) described the use of microscale purifi-

cation columns using RP resin.

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Kussman et al. J Mass Spec-

trom 32 (1997) 593-601.

Step 11: Microscale Desalting and Concentrating of Sample394

Tab. 11.1: Salt/buffer compatibility with MALDI analysis.

Type of impurity Concentration

Phosphate buffers <20 mmol/L

Tris buffer <50 mmol/L

Detergents <0.1%

Alkali metal salts <1 mol/L

Guanidine <1 mol/L

Ammonium bicarbonate <30 mmol/L

Glycerol To be avoided

SDS To be avoided

Sodium azide To be avoided

Microscale Desalting and Concentration

1. Take a gel-loader pipette tip and very carefully

pinch the tapered lower end of the tip with a

pair of flat armed tweezers.

2. Prepare a suspension of reversed phase resin in

methanol.

3. Add 50 mL of methanol to the pipette tip, fol-

lowed by 2–3 mL of the suspension.

4. A pipette can then be used to gently push the

methanol though the column. The RP resin

forms a small column at the end of the tip.

5. Equilibrate the column with 0.1% TFA (20 mL).

6. At this stage ensure the acetonitrile concentra-

tion of the peptide extracts is sufficiently low to

allow good retention of the peptides on the col-

umn. Hence dry the peptide extracts to a

volume of approximately 10 mL.

7. Further improve the retention of the peptide

mixture by acidifying the mixture with hepta-

fluorobutyric acid (HFBA). Add 9 mL of water

and 1 mL of HFBA to the semi-dried peptide

extracts, creating a 55% concentration (v:v) of

HFBA.

8. Load the acidified peptide extracts onto the col-

umn using a pipette. Gently push through the

solution with a pipette. Apply the eluate back to

the column; repeat five times to improve reten-

tion of the peptides.

Prevents the packing from

being eluted during preparation

and use.

Complete drying or lyophiliza-

tion not recommended, exten-

sive sample loss.

Heptafluorobutyric acid is a

very hydrophobic ion-pairing

reagent and will make the

digested peptides “very sticky”

improving there retention on a

reversed phase C18 column.

Step 11: Microscale Desalting and Concentrating of Sample 395

9. Wash the column with 0.1%TFA (20 mL).

10. Elute peptides with 3–5 mL of MeCN: 0.5% for-

mic acid (1:1, v:v).

Alternatively, these columns are available commercially (ZipTip

Follow the manufacturers instructions, which are similar to the

above.