1. Protein digestion and peptide mass fingerprint (PMF) analysis
Yuanming Luo
Institute of Microbiology,CAS

2. Protein digestion In gel digestion On-membrane digestion
In solution digestion

3. Two main approaches for digesting gel-separated proteins
Protein blotting onto immobilizing membranes followed by on-membrane digestion (simpson et al., 1989; Gevaert and Vandekerckhove 2000)
Digesting proteins directly in the gel matrix and extracting the peptides (Ward et al., 1990)

4. Note:
The overall recoveries of peptides from in-gel digestion methods are significantly greater than that from on-membrane digestion strategies.
In-gel digestion might fail when the visualized protein is below~10mg of protein/cm2 (need a further concentration of weakly stained CBB gel spots (membrane spots) with electrophoresis in a new gel).

5. In gel digestion
1. Excise the protein gel spots of interest and place in microfuge tube.
2. Remove excess CBB by washing twice with 1 ml of either 0.1 M NH4HCO3, 50% acetonitrile.
3. Dry each gel piece completely by speed Vac. The gel piece should not stick to the walls of the EP tube when completely dry.

6. In gel digestion (continued)
4. Rehydrate the gel piece by adding 10ul of digestion buffer, containing 0.5ug of the appropriate protease, directly onto the dried gel piece.
5. Store the gel pieces at 4℃ for over 45 min until the solution has been absorbed
6. If necessary, repeat above two steps to allow the gel pieces to fully swell.

7. In gel digestion (continued)
7. Add 20 ul of digestion buffer without protease to fully immerse the gel piece.
8. Incubate for hours at 37 ℃.
9. Carefully remove the digestion buffer (now called the extract), and place it into a clean microfuge tube.
The digestion buffer contains > 80% of the extractable peptides
10. Add 200 ul of 5 %TFA/50% acetonitrile to the gel piece.

8. In gel digestion (continued)
11. Incubate the tube with the gel piece for 1 hour at room temparature(37 ℃).
12. Carefully remove the extract away from the gel piece and combine it with previous extract from step 9.
Repeat step 10,11,12 once again.
Concentrate the pooled extracts by speed Vac. Store the peptide extracts at -20 ℃ for future mass spectrometric analysis.

9. In gel digestion of silver-stained gels
Most of the steps of in gel digestion for silver-stained gels are the same except for the destaining solution which was prepared by mixing 30 mM potassium ferricyanide with 100 mM sodium thiosulfate (1:1 v/v).

10. On-membrane proteolytic digestion of electroblotted proteins
Proposed membranes are nitrocellulose membranes, because the hydrophobic surface of PVDF limits the recovery of peptide fragments.
Proposed dyes are Amido Black and Ponceau S, which are compatible with proteolytic digestion, peptide extraction from membrane and subsequent RP-HPLC analysis of peptides.

11. Electroblotting of staining the protein
1. electroblot the proteins from the gel onto a nitrocellulose (for proteins that are difficult to transfer, add up to 0.005% SDS)
2. stain the membrane with either Amido Black or Ponceau S.
E.g., for staining with Amido Black
A. immerse the nitrocellulose membrane in 0.1% Amido Black 10B for 1-3 minutes
B. Rapidly destain with several washes of H2O/acetic acid/ methanol.

12. Electroblotting of staining the protein (continued)
C. Rinse the destained blots thoroughly with deionized H2O to remove any excess acetic acid.
D. cut out the stained protein band (or for 2D gel spots, up to 40 spots from identical gels may be required) and transfer these bands to 1.5-ml microfuge tubes for immediate processing (begin with step 3) or for storage at -20℃.

13. For staining with Ponceau S
A. Immerse the nitrocellulose membrane in 0.1% Ponceau S for 1 minute.
B. Gently agitate the blot for 1-3 min in 1% acetic acid to remove excess stain.
C. Cut out the protein bands of interest and transfer them to microfuge tubes.
D.Destain the protein bands by washing the membrane pieces with 200 mM NaOH for 1-2 min.
E. Wash the membrane pieces with deionized H2O and process them immediately or store them wet at -20℃(avoid excessive drying).

14. Digestion of the membrane-bound proteins
3. Add 1.2 ml of 0.5% (w/v) PVP-40 (which is used to prevent absorption of the protease to the nitrocellulose during digestion) in 100 mM acetic acid to each tube.
4. Incubate the tube for 30 min at 37 ℃.
5. Centrifuge the tube at ~1000g for 5 min.
6. Remove the supernatant solution and discard.
PVP, polyvinylyrrolidonne

15. 7. Add ~1ml of H2O to the tube.(It is essential to remove excess PVP-40 before peptide mapping because of the strong UV absorbance of this detergent. Moreover, breakdown products of PVP-40 produce major contaminant peaks in ESI-MS)
8. Votex the tube for 5 seconds.
9. repeat step 5 and 6.
10. repeat steps 7-9 five more times.
11. cut the nitrocellulose strips into small pieces (~1x1mm) and place them in a fresh tube (0.5- or a 0.2-ml tube).

16. Digestion of the membrane-bound protein (continued)
12. Add the minimal quantity of digestion buffer (10-20ml) to submerge the nitrocellulose pieces.
13. After digestion, tryptically 16 hours or overnight at 37 ℃, load the total reaction mixture onto an appropriate RP-HPLC column for peptide fractionation (or store the peptide mixture at -20 ℃ until use).

17. In soltion digestion

18. Mass spectrometry involved in proteomics
Matrix-associated laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)
Electrospray ionization (ESI) ion trap mass spetrometry
Surface-enhanced laser desorption ionization (SELDI) time of flight mass spetrometry (ProteinChip).

19. Linear and reflectron MALDI-TOF-MS
Linear MALDI-TOF-MS (lower accuracy)
Reflectron MALDI-TOF-MS: (1) higher accuracy, (2) Post-source decay (PSD), (3) delayed extraction

20. Mass spectrometry terms
Mass to charge (m/Z): Mass spectrometers measure the mass-to-charge values of molecular ions.
Resolution: resolution can be defined as the ability to separate and measure the masses of ions of similar, but not identical, molecular mass.
Signal to noise:
Monoisotopic mass versus average mass:

21. What is the difference between monoisotopic and average peptide mass?
As shown in the table, below the atoms that make up the naturally occurring amino acids found in proteins are not isotopically pure.
Natural Abundance of Isotopes Commonly Found in Proteins
Atom
Most Abundant Isotope
Next Most Abundant Isotope
Carbon
12C
98.9%
13C
1.11%
Nitrogen
14N
99.6%
15N
0.366%
Oxygen
16O
99.8%
18O
0.204%
Sulfur
32S
95.0%
34S
4.22%

22. Deisotoped MALDI spectrum

23. Interpreting of the MSn spectra of peptides (AGFI)

24. Singly-charged peptide fragmentation
Oxazolone,唑；carbonyl,羰基或碳酰基;amine,氨基

25. Reaction products: Proton on carbonyl (case 1)

26. Reaction products: Proton on amine (case 2)

27. Doubly-charged peptide fragmentation
Lys-C, triply charged

28. Reaction products: Proton on carbonyl (case 1)

29. Reaction products: Proton on amine (case 2)

30. Where is the b1 ion? Why is the a2/b2 big?

31. Peptide mass fingerprint (PMF) analysis by MALDI-TOF-MS

32. MALDI difficulties Salts and other contaminants Selection of matrix
Sample itself
Crystallization
Calibration (external or internal cablibration)
Accuracy (ppm)
Resolution
Amount of protease

33. Dissolve the peptide mixture in 0.1%TFA.
Desalt by ZipTip C18 microcolumn (optional).
Directly elute the peptide mixture with -cyano-4-hydroxy cinnamic acid (CHCA) in 70% acetonitrile and spot the peptide mixture on sample plate.
The peptide mixtures cocrystallize with matrix on sample plate

34. Input of instrumental method and parameter setup
Calibration of spectrometer by external calibration
Crude spectrum
Tuning of parameters
Internal calibration of PMF spectra
Data processing for database search

35. Peptide and protein standards
Angiotensin II (human)         MW: Substance P (human)            MW: Insulin (bovine)                     MW: Cytochrom c (equine)           MW: 12, RNase A (bovine)                 MW: 13, Apo-Myoglobin (equine)      MW: 16, Trypsinogen (bovine)           MW: 23,980.9

36. Manually spot samples onto sample plate

37. Dihydroxybenzoic acid,二羟苯甲酸

38. Recrystallization methods for MALDI matrices
Re-crystallization in 70% MeCN/30% water.
Heat a saturated solution of the matrix in 70% MeCN/30% water until boiling.
Carefully boil until solid dissolves completely.
Cool to room temp, then on ice - precipitate should form.
Filter precipitate.

39. Recrystallization methods for MALDI matrices
Protocol for re-crystallization of alpha-cyano-4-hyrdoxy-cinnamic acid
To 100mg of alpha-cyano-4-hydroxy-cinnamic acid, add 10ml of water
Add ammonium hydroxide until most of acid dissolves.
Slowly add concentrated HCl to the solution until a large amount of the acid has precipitated (about pH 2).
Remove the precipitate by centrifugation or filtering.
Wash the precipitate several times with 0.1M HCl.
Dry matrix and store at -20 C in the dark.

40. Calibration
Used for calibration

42. It would be extremely difficult to measure a monoisotopic mass for BSA
It would be extremely difficult to measure a monoisotopic mass for BSA. In practice, most instruments report monoisotopic molecular weights up to a certain cut-off point. Above this cut-off, isotopic envelopes are centroided as a whole to provide average mass values.

43. Control the sample plate
CHCA

44. Parameters setup of PMF
Instrument：Voyager DETM PRO BiospectrometryTM workstation,
Parameters： matrix:α-Cyano-4-hydroxycinnamic acid(CHCA) (1)delayed extraction, 150 ns、(2)reflector mode、(3)positive; (3)acceleration voltage: V；(4)Grid voltage75%；(5)Guide wire: 0.02%；(5)mass range: Da。

45. PMF containing isotope peaks

46. monoisotope spectrum

47. PMF spectrum of annexin I

48. Mass list of annexin I (monoisotope)

50. Database search by MASCOT

56. Key points for PMF Sample preparation
Matrix selection and ratio of sample to matrix
Keep the matrix dry
Sample properties
Internal calibration
Database selection
Mass accuracy (ppm)
Resolution (FWHM) and S/N when tuning

57. Practical consideration
-The final molar ratio sample/matrix is about or around 1/5000.
-Final concentration of the sample is from 1 to 10 pmol/ul
-Our experience with MALDI point to a dynamic range of 100 fmol/ul to few hundreds pmol/ul
-MALDI is relatively robust ionization technique that tolerates the use of salts and surfactants and buffers. Although it is best to remove them for better performance.

58. Theoretical digestion for PTM identification analysis

60. Application of MALDI-TOF-MS
Molecular weight determination
Peptide mass fingerprint
Disulfide bond identification
C-terminus sequencing