Protein Sequencing

Protein Sequencing

 Permits comparisons between normal and mutant proteins.  Permits comparisons between comparable proteins in different sps.  Vital piece of information for determining the 3-D structure of a prote

Why are peptides, and not proteins, sequenced? Primary structure of proteins is determined.

  

Solubility under the same conditions Sensitivity of MS much higher for peptides MS efficiency

Sequencing of Peptides

ut the order of amino acids in a peptide, sequential remova tification of residues from one or the other free terminal of peptide chain is carried out.

es a problem: long polypeptides contamination of amino acids removed.

the solution: Polypeptide chain is broken into short sequence Short sequences reassembled to obtain overall seq.

tep I– Purification of protein

ifferential Precipitation with (NH4)SO4 olumn Chromatography el Electrophoresis ifferential Centrifugation

Main steps: Purification of a protein Cleavage of all disulfide bonds Determination of the terminal amino acid residues Specific cleavage of the polypeptide chain into small fragments Independent separation and sequence

Step II--Cleavage of disulfide bonds

Step III--Determination of polypeptide-chain end grou Amino terminal--FDNB

Dansyl Chloride Method

terminal—Anhydrous Hydrazine at 100ºC amino acid hydrazides except for all erminus ones (free acid) and this can be identified by chromatography.

Treat polypeptides with carboxypeptidase. Results in release of carboxy amino acid as the major free amino acid which is identified chromatographically

III—Fragmentation of peptide chain into short sequ A. Use of endopeptidases

Choice of Enzyme Cleaving agent/Proteases A. HIGHLY SPECIFIC Trypsin Endoproteinase GluC Endoproteinase LysC Endoproteinase ArgC Endoproteinase AspN B. NONSPECIFIC Chymotrypsin Thermolysin

Specificity Arg-X, Lys-X Glu-X Lys-X Arg-X X-Asp Phe-X, Tyr-X, Trp-X, Leu-X X-Phe, X-Leu, X-Ile, X-Met, XVal, X-Ala

B. Use of Cyanogen bromide

ific cleavage at Met residue. version of free carboxyl-terminal Met to Homoserine lactone.

Step IV--Peptide Analysis  

Edman Degradation MS (Mass Spectrometry)   



More sensitive Can fragment peptides faster Does not require proteins or peptides to be purified to homogeneity Has no problem identifying blocked or modified proteins

Edman degradation Phenyl isothiocyanate H O N C

S

+

H2N C C

O Asp

Phe

Phe

Arg

C O

CH3

-

Labeling

H

S

H H O

N C N C

C

O Asp

Phe

Phe

Arg

C

CH3

O-

Release S N

O CH3

N O

+

H2N Asp

Phe

Phe

Arg

C

H

PTH-alanine

O-

Peptide shorthened by one residue

Treatment of a N­unprotected peptide with PITC (C6H5N=C=S) followed by acid.  It  allows for the successive “stripping away” of the N­terminal amino acid in the form  of  a  phenylthiohydantoin.    In  the  process,  it  produces  a  new  shortened  peptide  with a new, exposed N­terminus. PTH amino acid is identified by TLC 

Edman Degradation vs. MS/MS

s Involved in Protein Identification and Annotation

ein Samples olve protein mixtures by 2-D gel electrophoresis Chromatography

vidual Proteins estion by enzymes or mass spectrometry MS Fragment Ion Analysis DI-TOF Peptide Mass Fingerprinting

abase Search

Tandem Mass Spectrometry 



  

MS used for accurately determining molecular masses by calculating mass:charge ratio of ions in a vacuum Combines an instrument for source of ions, mass analyser to separate ions by mass:charge ratio and an ion detector MS/MS plays important role in protein identification (fast and sensitive). Derivation of peptide sequence an important task in proteomics. Derivation without help from a protein database (“de novo sequencing”), especially important in identification of unknown protein.

Basic lab experimental steps 1. Proteins digested w/ an enzyme to produce peptides 2. Peptides charged (ionized) by MS and separated according to their different m/z ratios 3. Each peptide fragmented into ions and m/z values of fragment ions are measured 

Steps 2 and 3 performed within a tandem mass spectrometer.

Mass spectrum 

Proteins consist of 20 different types of a. a. with different masses (except for one pair Leu and Ile)



Different peptides produce different spectra



Use the spectrum of a peptide to determine its sequence

MS Peptide Experiment

MALDI (matrix-assisted laser desorption ionization) 3 nS LASER PULSE

+++ + + + ++ + ++ + + ++ + ++ + ++ ++

Sample (solid) on target at high voltage/ high vacuum

+ + ++ + + + + ++ + ++ ++ + +

+ ++ + ++ + + ++ ++ ++ + +++ + ++ ++ +++ ++ +

+ + +++ ++ + ++ +++ + ++ +++++ + +++ +++ +++ + +

TOF analyzer High vacuum

MALDI is a solid-state technique that gives ions in pulses, best suited to time-of-flight MS.

ization method for peptide mass. fragments mixed with light-absorbing matrix compound (DHBA) in an organic so evaporated to form crystals, transferred to vacuum with a laser beam. nergy absorbed and emitted (desorbed) as heat. ion of matrix and anylation into gas phase. ltage applied across the sample to ionize it and ions are accelerated towards the

ESI (Electrospray Ionization) Liquid flow

Q or Ion Trap analyzer

Atmosphere Low vac. High vac.

ESI is a solution technique that gives a continuous stream of ions, best for quadrupoles, ion traps, etc.

ization method used for ion searching is dissolved in a solvent, pushed through a narrow capillary al difference applied across the capillary such that charged droplets eme fine spray. of heated inert gas applied and each droplet evaporates. enters the mass analyzer and ions are accelerated towards the detecto

….MALDI or Electrospray ? MALDI is limited to solid state, ESI to liquid ESI is better for the analysis of complex mixture as it is directly interfaced to a separation techniques (i.e. HPLC or CE) MALDI is more “flexible” (MW from 200 to 400,000 Da)

Protein Identification Strategy *

I 12

Peptides

Protein mixture

14 Time (min)

16

1D, 2D, 3D peptide separation 10-Mar-200514:28:10

II

CAL050310A 71 (1.353) Cm (1:96)

*

TOF MSMS 785.60ES+ 2.94e3

684.17

100

333.15

813.16

480.16

785.62

%

942.16

685.18 187.07 246.13 175.12

1171.14 943.17

740.09

627.17 612.08 498.09

286.11 382.11

1285.14

1056.17

814.17

497.09

1057.18

200 400 600 80010001200 480.08

m/z

169.06

Q1

Q2 Collision Cell

10-Mar-200514:28:10 CAL050310A 71 (1.353) Cm (1:96)

Q3

III

813.16

480.16

685.18 187.07 246.13 175.12 286.11 382.11

100

942.16

480.08

627.17 612.08 498.09

1285.14

1056.17

814.17

1171.14

497.09

169.06

0

Correlative sequence database searching

785.62

%

943.17

740.09 924.16

1057.18

1039.13 1058.17

1286.14 1172.15

1173.16

CAL050310A 71 (1.353) Cm (1:96)

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1600

m/z

200 400 600 80010001200

600

700

800

900

1000

1100

785.62

286.11 382.11

100

942.16

480.08

627.17 612.08 498.09

1285.14

1056.17

814.17

1171.14

497.09

943.17

740.09 924.16

1057.18

1039.13 1058.17

1286.14 1172.15

1173.16

200

300

400

500

600

700

800

900

1000

1100

1287.13 1296.10

1038.17

1200

1300

1400

1500

1600

m/z

200 400 600 80010001200

m/z

Theoretical

500

813.16

685.18

169.06

1500

400

TOF MSMS 785.60ES+ 2.94e3

480.16

187.07 246.13 175.12

0 1400

300

684.17

%

1287.13 1296.10

1038.17

200

1039.13 1058.17

m/z

Protein identification

1173.16

1287.13 1296.10

1038.17

1200

1300

1400

1500

Tandem mass spectrum 333.15

333.15

100

924.16

10-Mar-200514:28:10 100

TOF MSMS 785.60ES+ 2.94e3

684.17

100

0

1286.14 1172.15

Acquired

1600

m/z

Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry, Molecular & Cellular Proteomics, 2003, 2.11, 1234, Thomas S. Nuhse, Allan Stensballe, Ole N. Jensen, and Scott C. Peck

What you need for peptide mass mapping 

Peptide mass spectrum



Protein Database 



GenBank, Swiss-Prot, dbEST, etc.

Search engines 

MasCot, Prospector, Sequest, etc.

Protein Identification by MS

Library

Spot removed from gel

Artificial spectra built

Fragmented using trypsin

Spectrum of fragments generated

MATC H

Artificially trypsinated

Database of sequences (i.e. SwissProt)

Conclusions 



MS of peptides enables high throughput identification and characterization of proteins in biological systems “de novo sequencing” can be used to identify unknown proteins not found in protein databases