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

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397

Step 12: Chemical Derivatization of the Peptide Digest

12.1

Guanidation and Sulfonation of the Peptide Digest

This method (Keough et al. 1999; 2000a 2000b) describes the proce-

dure for the guanidation of lysine residues and the subsequent sulfo-

nation of the a-amino group at the N-terminus of peptides in the

digest mixture. The sulfonation reagent, 2-chlorosulfonyl acetylchlor-

ide is highly hygroscopic and strenuous precautions must be made to

ensure that water (including moisture in the atmosphere) is absent

from the reagents and reaction vessel.

1. Take combined peptide extracts and concentrate

in a speed vac to a volume of 20 mL.

2. Guanidate the lysine side chain. Mix 2mL O-

methylisoureahydrogen sulfate solution (86

mg/mL MilliQ H

O) with 8 mL 0.25 mol/L

NaHCO

, pH 11.5 and add to digest. Place it in

the oven at 37 C for 2h. (The reaction can also

be performed at room temperature overnight or

at 70 C for 10 minutes).

3. Remove reagents using a reversed phase micro-

scale desalting and concentration column as

described in Step 9 above, elute peptide digest

and evaporate solvent to dryness.

4. Reconstitute in 10 mL THF:DIEA 19:1.

5. Add 2 mL sulfonation reagent (2 mL neat mate-

rial in 1 mL THF)

6. React for 1–2 min. at room temperature.

7. Dry completely to remove organics and excess

base.

8. Reconstitute in 5–10 mL of 0.1% TFA.

9. Apply to MS target as described in Step 11. 2,5

DHB is the preferred matrix of choice, though

a-cyano 4 hydroxy cinnamic acid can be used.

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 12: Chemical Derivatization of the Peptide Digest398

12.2

Sulfonation of the Peptide Digest Using CAF Chemistry

This method describes sulfonation of the a-amino group at the n-ter-

minus of peptides in the digest using a novel, modified water-stable

reagent. Steps 2 and 3 can be omitted if guanidation of the lysine

groups in the peptide digest is not favored.

1. Apply peptide digest extracts to a reversed phase

microscale desalting and concentration column

as described in Step 9 and dry down extract to a

minimal volume. Alternatively the peptide can

be dried down to a minimal volume and pro-

ceed from step 2 (or step 4 if the guanidation

step is to be omitted).

2. Reconstitute peptide digest in the guanidation

reagent [mix 2 mL O-methylisoureahydrogen

sulfate solution (86 mg/mL MilliQ H

O) with

8 mL 0.25 mol/L NaHCO

, pH 11.5].

3. Place it in 37 C for 2 h. (The reaction can also

be performed at room temperature overnight or

at 70 C for 10 minutes)

4. Prepare fresh reagent. Dissolve the CAF reagent

in 0.25 M NaHCO

, pH 9.4 (10mg/100 mL).

5. Apply 10 mL of the CAF-reagent solution to the

sample and leave for 15 minutes.

6. Add 1 mL 50% hydroxylamine solution. Reverses

tyrosine modifications at the hydroxyl group.

7. Add 8 mL water and 1 mL HFBA to the digest

solution and apply to a reversed phase C18 col-

umn as described in section Step 9

8. Elute derivatized peptides in aceonitrile: 0.5%

formic acid (1:1, v:v) 1–5 mL

9. Apply to the MALDI target as described in

Section 13.1.

399

Step 13: MS Analysis

13.1

Sample Preparation for MALDI

Three matrices are generally used for peptide and protein analysis. A

variety of methods have been described, particularly for the use of

a-cyano-4-hydroxy-cinnamic acid for peptide analysis. Notably, Vorm

et al. described the thin film method and Jensen et al. 1996 incorpo-

rated nitrocellulose into a similar method for improved performance.

This section will describe the dried droplet method, probably first

choice for most applications for each of the three matrices concerned.

It is important in each case to keep the size of the target spot as small

as possible. Remember the width of the laser beam is very narrow

(~20 microns) and as such the majority of the sample will not be

ablated with the laser from a large sample spot.

& Do not use non-volatile solvents (e.g. glycerol,

polyethyleneglycol, DMSO, Triton X or

2-mercaptoethanol) with this method.

13.2

a-Cyano-4-hydroxy-cinnamic acid. Analysis of Peptides and Peptide

Digests

1. Prepare a fresh solution of a-cyano-4-hydroxy-

cinnamic acid in acetonitrile:0.1 %TFA (1:1, v:v;

10 mg/mL). Prepare a fresh solution of matrix

wherever possible.

2. Take 0.2–0.5 mL matrix and mix with 0.2–0.5 mL

of sample. Mix thoroughly by uptaking and dis-

placing the volume from a pipette several times.

For reproducible analysis, thorough mixing of

the matrix and analyte is necessary.

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

Vorm O, Roepstorff P, Mann

M. Anal Chem 66 (1994)

3281-3287.

Jensen ON, Podtelejnikov A,

Mann M. Rapid Commun

Mass Spectrom 10 (1996)

1371–1378.

Loading the sample in high

organic content (high methanol

or acetonitrile causes the

droplet to spread uncontrollably

across the target surface.

It is important to keep the pH

of the matrix solution below pH

4, using 0.1% TFA maintains

and ensures this.

If the analyte concentration is

sufficiently large then 1–2 mL

can be mixed with several mLof

matrix and subsequently

vortexed before applying to the

target.

Step 13: MS Analysis400

3. Apply to the target and leave to dry at room tem-

perature.

4. Insert target into mass spectrometer.

13.3

2,5 Dihydroxybenzoic Acid. Analysis of Peptide Digests

1. Prepare a solution of 2,5-dihydroxybenzoic acid

in 0.1% TFA (10mg/mL).

2. Take 0.2–0.5 mL matrix and mix with 0.2–0.5 mL

of sample. Mix thoroughly by uptaking and dis-

placing the volume from a pipette several times.

For reproducible analysis, thorough mixing of

the matrix and analyte is necessary.

3. Apply to the target and leave to dry.

4. Insert target into mass spectrometer.

2,5-Dihydroxybenzoic acid is commonly used for the analysis of oligo-

saccharides released from glycoproteins, the above method can be fol-

lowed by the analysis of oligosaccharides.

Tip – oligosaccharide analysis Since 2,5-DHB forms long needle-like

crystals, homogeneity of the sample can be increased by redissolving

the dried matrix-sample with ethanol. Apply 0.1 mL of ethanol to the

dried spot.

13.4

Sinapinic Acid. Analysis of Proteins

1. Prepare a solution of sinapinic acid in acetoni-

trile and 0.1% TFA (60:40, v:v; 10mg/mL).

2. Take 0.2–0.5 mL matrix and mix with 0.2–0.5 mL

of sample. Mix thoroughly by uptaking and dis-

placing the volume from a pipette several times.

For reproducible analysis, thorough mixing of

the matrix and analyte is necessary.

3. Apply to the target and leave to dry.

4. Insert target into mass spectrometer.

Do not heat the sample target

to speed up drying process

(place on a hot plate).

The concentration of acetoni-

trile can be increased up to

30%.

Dried droplets are quite stable;

they can be kept in a drawer or

in vacuum for days.

13.5 Sample Preparation for ESI MS 401

13.5

Sample Preparation for ESI MS

Nanospray

In electrospray mass spectrometry the presence of salts,

involatile buffers and polymeric material in a sample is often detri-

mental to the analysis. High amount of salts often result in partial or

complete blocking of the needle orifice resulting with, at best, an

intermittent and instable spray. Subsequently, sensitivity is reduced

and the spectrum can be complicated with salt adducts. The ingredi-

ents in a protein digest reaction, salts and possibly detergents, are

present in millimolar amounts and they have to be removed before

analysis. As with MALDI analysis, a microscale desalting and concen-

tration step often eliminates this problem and at the same time

allows a concentration of the analyte(s).

A microscale desalting and purification column is prepared as

explained in step 9. Peptides are eluted into the nanospray needle

using 50% methanol:0.5% formic acid, or eluted into a sample tube

and pipetted into a nanospray needle.

LC-MS/MS Following the in-gel digestion step redissolve the digest

extract in 5 mL water. (Optional, 0.5 mL can be used for MALDI MS

analysis). The remaining 4.5 mL is mixed with 4.5 mL of a 0.1% aqu-

eous formic acid solution for subsequent ESI LC-MS/MS analysis.

The remainder of the digest extract solution can be loaded on an auto-

sampler, from which 5 mL are injected into a nano-HPLC (or alterna-

tively manually injected). Separation is typically performed using a

75 mm  150 mm silica C18 (3 mm particle size) PepMap column

(LCPackings, Amsterdam, The Netherlands) with 0.1% formic acid in

water as solvent A and 0.08% formic acid in 80% acetonitrile / 20%

water as solvent B by starting after equilibration with 5% solvent B

linearly increasing it to 40% within 32 min using a flow rate of

200 nL/min. The nano-HPLC is directly coupled to a tandem mass

spectrometer equipped with electrospray ionization. This method

was supplied by Dr Rainer Cramer, Ludwig Institute for Cancer

Research.

A microscale desalting and

concentration may be less

successful for removing deter-

gents.

403

Step 14: Calibration of MALDI-ToF MS

The MALDI peptide mass fingerprint can be calibrated internally or

externally.

14.1

Internal Calibration for Peptide Analysis

Trypsin autolysis during the digestion of the target protein(s) can be

very useful for calibration purposes. However, it is important to know

which species the trypsin is derived from, porcine or bovine. It is for-

tunate that the two major porcine trypsin autolysis peaks present in

the peptide mass fingerprint actually bracket the majority of the mass

range of interest. The internal calibration procedure will be depen-

dent on the MS in use, but the suggested two peptides to use are:

Trypsin II 842.509 [M+H]

Trypsin IV 2211.104 [M+H]

Both of these molecular weights are the monoisotopic C12 isotope,

protonated form of each peptide. Other porcine trypsin autolysis

peaks observed in a MALDI PMF include 515.32, 1045.56, 2283.17,

and 2299.17 (2283.17 with oxidized Met).

Using an internal calibration, high-mass accuracy is achievable

(sub 25 ppm).

The expected peptides from a theoretical trypsin digest of porcine

and bovine trypsin are listed in Tables 14.1 and 14.2.

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 14: Calibration of MALDI-ToF MS404

Tab. 14.1: Theoretical tryptic peptides of porcine trypsin, TRYP_PIG

(protonated monoisotopic

C mass, unprotonated average mass).

Position in

sequence

Monoisotopic

mass [M+H]

Average

mass [M]

Sequence

52–53 262.14 261.28 SR

54–57 515.32 514.63 IQVR

108–115 842.50 842.01 VATVSLPR

209–216 906.50 906.05 NKPGVYTK

148–157 1006.48 1006.15 APVLSDSSCK

98–107 1045.56 1045.16 LSSPATLNSR

134–147 1469.72 1469.68 SSGSSYPSLLQCLK

217–231 1736.84 1736.97 VCNYVNWIQQTIAAN

116–133 1768.79 1768.99 SCAAAGTECLISGWGNTK

158–178 2158.02 2158.48 SSYPGQITGNMICVGFLEGGK

58–77 2211.10 2211.42 LGEHNIDVLEGNEQFINAAK

78–97 2283.17 2283.63 IITHPNFNGNTLDNDIMLIK

179–208 3013.32 3014.33 DSCQGDSGG...SWGYGCAQK

9–51 4475.09 4477.04 IVGGYTCAA...VVSAAHCYK

9–51 4489.11 4491.07 IVGGYTCAA...VVSAAHCYK

Tab. 14.2: Theoretical tryptic peptides of bovine trypsin, TRY1_BOVIN

(protonated monoisotopic

C mass, unprotonated average mass).

Position in

sequence

Monoisotopic

mass [M+H]

Average

mass [M]

Sequence

110–111

260.19 259.35 LK

157–159 363.20 362.49 CLK

238–243 633.31 632.67 QTIASN

64–69 659.38 658.76 SGIQVR

112–119 805.41 804.86 SAASLNSR

221–228 906.50 906.05 NKPGVYTK

160–169 1020.50 1020.17 APILSDSSCK

229–228 1111.55 1111.33 VCNYVSWIK

146–156 1153.57 1153.25 SSGTSYPDVLK

207–220 1433.71 1433.65 LQGIVSWGSGCAQK

191–206 1495.61 1495.61 DSCQGDSGGPVVCSGK

70–89 2163.05 2163.33 LGEDNINVVEGNEQFISASK

170–190 2193.99 2194.47 SAYPGQITSNMFCAGYLEGGK

90–109 2273.15 2273.60 SIVHPSYNSNTLNNDIMLIK

120–145 2552.24 2552.91 VASISLPTS...LISGWGNTK

21–63 4551.12 4553.14 IVGGYTCGA...VVSAAHCYK

Obtained with permission

from: www.matrixscience.com.

Obtained with permission

from: www.matrixscience.com.

14.2 External Calibration for Peptide Analysis 405

The practice of adding synthetic peptides to the peptide digest to

perform an internal calibration is not recommended for two reasons.

First, it is difficult to estimate the concentration of calibrants to add;

too high concentration may suppress the ionization of the peptides of

interest. Second, the calibrants may co-incide with peptides of inter-

est, therefore precluding these peptides from the subsequent data-

base search.

14.2

External Calibration for Peptide Analysis

When one or both of the autolysis peaks are absent from the spec-

trum, an external calibration has to be performed. This is normally

achieved by calibrating a spectrum acquired from the sample spot

adjacent to the sample spot whose spectrum needs to be calibrated.

Hence it is often useful to have a calibration sample alongside each

sample, or at least every two samples. Hence an internal calibration is

performed on the calibration mixture, the calibration file saved and

then applied to the spectrum of interest (the procedure will depend

upon the MS instrument used). This is a useful fallback method in

case successive spectra cannot be calibrated internally from the tryp-

sin autolysis peaks.

As opposed to the internal calibration where only two autolysis

peaks act are used as the calibrants, the external calibration mixture

can contain several calibrants defining the calibration curve more effi-

ciently. A table of potential calibrants is listed below.

Tab. 14.3: Suggested peptides for use in an external calibration mixture

(All molecular weights are

C monoisotopic values).

Peptide Monoisotopic mass [M+H]

Angiotensin I 1296.678

Angiotensin II 1046.535

Substance P 1347.728

[Glu]-fibrinogen b 1570.670

Renin substrate 1759.932

Apamin 2026.887

HACTH clip 18–39 2465.188

HACTH clip 7–38 3657.922

& Each combination of instrument mode (detection

mode (linear/reflectron), accelerating potential)

needs individual calibrations.

Obviously a calibration file

obtained from the internal cali-

bration using both trypsin auto-

lysis peaks can be applied to

the spectrum acquired from an

adjacent spot, without having

to use a calibration mixture

such as this.

407

Step 15: Preparing for a Database Search

This section describes the function of each category observed on a

commonly used search engine web browser for peptide mass finger-

printing and peptide sequence analysis.

15.1

Number of Missed Cleavages

The number to be input is dependent on the efficiency of the diges-

tion. If the digestion is ideal then the enzyme cleaves at the correct

site every time, affording the highest discrimination. However,

enzymes rarely act in such an ideal fashion and partial peptides can

be observed in the peptide mass fingerprint. Typically, one missed

cleavage is selected as this. Selecting a higher number than this can

reduce discrimination, as the experimental peptide mass list will be

searched against more theoretical peptides.

15.2

Choice of Cleavage Agent

Table 1.8 in Section 1.7.2.1 lists a range of enzymes available for pro-

teolytic digestion. However, not all of the listed enzymes are suitable

for subsequent protein ID. Enzymes with low specificity, particularly

those generating small peptides should be avoided. Trypsin is the

most commonly used enzyme as it yields peptides across a suitable

mass range, relatively high specificity and locates the basic residues

at the c-terminus of the peptide. This is useful for subsequent MS

and MS/MS analysis.

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 15: Preparing for a Database Search408

15.2.1

Choice of Amino Acid Modification

Proteins can be modified as a result of cellular processes, specifically

post-translational modications; a sample preparation, including

methionine oxidation and/or directly as part of an analytical method

such as the reduction and alkylation of cysteine residues.

These modifications can be regarded as complete or partial for a

database search. For instance, if there is uncertainty about the quanti-

tative modification of a particular residue, or whether a modification

of a particular residue exists at all then the partial modification option

can be used. In this instance, the search engine uses the masses of

both the native amino acid and the modified amino acid in the

search. For example, when a protein is applied to the second dimen-

sion the methionine residues present in the protein are prone to oxi-

dation due to the conditions of electrophoresis. Oxidation of a

methionine residue results in a mass increase of 16 Da (nominal

mass). Therefore, selecting a partial modification of the methionine

enables both forms to be identified if they are present.

Second, if all cysteine disulfide bonds were quantitatively reduced

and the resulting cysteine residues alkylated with iodoacetamide prior

to the second dimension, and the procedure repeated prior to in gel-

digestion then theoretically all cysteine amino acids will now have a

mass of 160 Da and not 103 Da (nominal mass). For a database

search, the search engine requires this information if it is to correctly

identify any cysteine containing peptides.

It is very difficult to predict the presence of post-translational mod-

ifications. A particular example is phosphorylation. It would be

unwise to simply use a partial phosphorylation calculation on all the

potential residues.

15.3

Peptide Mass Fingerprint

This is where the masses from the PMF are manually inputted. A

trypsin digestion yields useful peptides in the 800–3500 Da mass

range. Select peptides that fall within this mass range. Using masses

much below this range is unadvisable as they be matrix related or too

small to be significant in the search. Further, Fenyo et al. (1998)

demonstrated that a high mass peptide was useful for constraining

the database. The number of peptides submitted to the search should

also be considered. Too many peptides can be as problematic as too

few peptides (see Figure 3.16 in Section 3.3.3). Peptide masses which

cannot be matched with the correct protein contribute to the num-