PMF Procedure

PROCEDURE FOR MAKING PEPTIDE MASS FINGERPRINTS TO IDENTIFY PROTEINS

The procedure below for preparing samples for peptide mass fingerprinting has two variations. One is for samples of parchment, hide, gut material, etc., where the sample is mainly protein.  The other is for samples from artworks, such as paint, ground, binder, etc., where the protein may be in low concentration and entrained in a solid matrix. The main difference is that the solubilization steps for paint samples are more aggressive. Additional information can be found in the papers listed in the References section of the blog.

GENERAL CONSIDERATIONS: Collect samples into 600 µL Eppendorf tubes or equivalent, if possible. Excise micro samples from objects, paintings, etc., paying particular attention to avoid contaminating the sample with proteins such as keratins (skin, dust, saliva). Use a clean, sharp scalpel, tweezers, or a sharp needle. Clean tools with ethanol or other solvent between samples.  Avoid using a fine brush for sampling as it is easily contaminated. If it is necessary to use a fine brush, for example to transfer staticky particles, clean the brush carefully between samples and inspect it under the microscope. Transfer samples from microscope slides under a microscope to be sure the sample gets into the digestion tube. 600 µL tubes (or smaller) are preferred for digestion because they allow the small liquid volumes to vortex high up the tube sides to capture particles into the liquid.  CAUTION: Static on the tube can cause samples to fly away.

Work in a clean hood whenever sample tubes are opened to add reagents.

 

SAMPLE is:

Parchment, hide, gut (mainly collagen).

SAMPLE is:

Paint with pigment, binder or ground.

SOLUBILIZATION

1. Add 60 µL 50 mM AMBI (ammonium bicarbonate) to samples; vortex (~5 s) and spin in centrifuge (~15 s).

1. Add 30 µL TFE (2,2,2-trifluoroethanol); vortex (~5 s) and spin in centrifuge (~15 s).

 

2. Add 30 µL 50 mM AMBI (ammonium bicarbonate) to samples; vortex and spin.

Check samples under magnification to be sure they are in the liquid. If not, flick down solid on vial walls. The entire sample must be in the liquid in order for the method to be successful.

3. Heat at 80°C for 60 min.; vortex and spin samples 2-3 times during this period; check that samples are in the AMBI solution.

3. Heat at 60°C for 30 min.; vortex and spin samples every 10 min.; check that samples are in the TFE/AMBI solution.

 

4. Ultrasonicate samples for 10 min.; check samples individually to be sure each is completely disrupted.

 

5. Heat samples at 60°C for 10 min., vortex and spin; check that samples are in the liquid; mark the liquid level on the tube.

 

6. Process samples in the vacuum centrifuge at 30°C for 15 min. Repeat as necessary until volume is <20 µL. Settings: V-AQ (vacuum, aqueous) Temperature: 30°C.

 

7. Add 30 µl 50 mM AMBI; heat at 60°C for 30 min.; vortex and spin every 10 min.

 

 

REDUCTION AND ALKYLATION

8. Cool samples to RT. Add 3 µL 20 mM TCEP (tris (2-carboxyethyl) phosphine hydrochloride) in 25 mM AMBI. Heat to 37°C for 20 min.

9. Add 3 µL 40 mM IAA (iodoacetamide) in 25 mM AMBI. Allow to react 30 min., RT, dark.

TRYPTIC DIGESTION

10. Add 8 µL trypsin (0.02 ug/µL in 50 mM AMBI). Digest at 37°C at least 2 hr. but preferably overnight.

MATRIX AND SAMPLE PREPARATION FOR MALDI ANALYSIS

11. Add 1 uL formic acid or 0.1% (v/v) TFA; vortex and spin solids to bottom of tube. Vacuum centrifuge as necessary to reduce volume.

12 Prepare Matrix:  Saturated CHCA  (α-cyano-4-hydroxycinnamic acid) in 40% (v/v) ACN (Acetonitrile) 0.1% (v/v) TFA (trifluoroacetic acid). Add a small amount of CHCA to a glass vial, add 40% ACN 0.1% TFA, vortex intermittently for 5 minutes and allow undissolved CHCA to settle.

13. Prepare samples: Combine 20 µL matrix and 2-3 µL sample in a new 600 µL tube; Vortex and spin. Spot ~1.5 µL onto the MALDI plate; allow to air dry. Do not allow pipette tip to touch the MALDI plate as this interferes with proper matrix crystallization. Check dried spots under the microscope to observe proper crystallization.

 

When samples are dried on the sample plate, they are stable for at least several weeks if kept from light and can be transported to available equipment when convenient.

The equipment and reagents described below are typical for this type of analysis, and substitutions can be made. If there is any doubt about specific equipment, reagents or procedures, a positive control, such as lab gelatin, can be used to validate the method. A reference spectrum for lab gelatin (bovine) is included here for comparison.  ## Not required for parchment/collagen samples.

 Please see the end of this page for a reference MALDI peptide mass fingerprint for lab gelatin from a bovine source.

Digestion consumables

Company: VWR

Item

VWR Cat. #

Trifluoroacetic acid, EMD Chemicals, 100  g

EM-TX1275-1

Formic acid AR* ACS Grade, 120 mL

MK259202

Ammonium bicarbonate, Baker Analyzed Reagent, 500 g

JT3003-1

pH Indicator strip, EMD, 0-14, pkg 100

EM-9590-3

Pipet tips, Gilson-Style, Axygen No. RFL-300-C, 0.5-10 uL, cs of 5

89029-921

Pipet tips, Universal Fit, Axygen No. RFL-222-C, 1-200 uL, pack of 960

89029-919

Serapettor bottletop dispenser 1-10  mL BrandTech No. 4721140

18910-218

Microcentrifuge tubes, 0.65 mL, natural,  pack of 1000

89000-010

Microcentrifuge tubes, 1.5 mL, natureal, pack of 500

89000-028

Eppendorf gel loading tips pk 192

 53511-646

Eppendorf Cat. # 022351656

Company: Sigma Aldrich

Item

Sigma Aldrich Number

Acetonitrile, Chromsolve Plus >99.9%, 1L

34998-1L

HPLC water, Chromasolve, 4L

270733-4L

Methanol, Chromasolve, >99.9%, 2L

34860-2L-R

##2,2,2 trifluoroethanol, Fluka, 100 mL

91690-100 mL

Conical centrifuge tubes, 15 mL, pkg 100

CLS430055-100ea

Iodoacetamide 5 g

I6125-5G

alpha-cyano-4-hydroxycimmamic acid, >=99% (CHCA)

Sigma-Aldrich #70990-1G-F

Company: Promega

Item

Promega Number

Promega, Sequencing grade modified trypsin, 100 ug, lyophilized, Customer Service        1-800-356-9526

V5111

 

Typical equipment used for protein digests

 

 

VWR Cat. #

Micro centrifuge,  VWR Mini

37000-700

Vortex Mixer, VWR Signature, Digital

14005-824

Thermomixer, Eppendorf # 022670000

21516-170

##Ultrasonic bath, VWR Symphony

97043-964

##Eppendorf 5305 Vacufuge (Eppendorf Cat. #22820109)

53512-624

Freezer, under counter, Northland-Marvel 4.5 cu ft

VF-4CAF

*Bench top clean hood, Filtco, dist. by Air Science USA

*VLF-24

Analytical Balance, Ohaus #AV114C

87000-802

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