Characterization of Proteins

Determination of the Sizes of Proteins

• Sedimentation analysis

• Centrifugation• Gel filtration• SDS PAGE

Differential Precipitation

• Precipitation:

• The process of formation of a solid that was previously held in  solution. 

– NH4 

SO4 

– Polyethylene glycol are added to a protein solution

• A precipitate forms and separated from the solution after  centrifugation.

• If the concentrations of NH4 

SO4 

or polyethylene glycol are  increased, more precipitate forms. 

3

Centrifugation

• Zonal centrifugation:  Mixture to be separated is layered on top of a gradient (e.g.

sucrose or ficoll) increasing concentration down the tube ‐

can be continuous or 

discontinuous (layers)

‐

provides gravitational stability as different species move down

tube at different 

rates forming separate bands. 

– Species are separated by differences in SEDIMENTATION COEFFICIENT

(S) 

= 

Rate

of movement down tube/Centrifugal force

– S is increased for particle of LARGER MASS

(because sedimenting force a M(1‐vr)

– S is also increased for MORE COMPACT STRUCTURES of equal particle mass 

(frictional coefficient is less)

Zonal ultracentrifugation

Centrifugation

• Isopycnic (equal density) centrifugation: Molecules separated on 

EQUILIBRIUM POSITION, NOT by RATES of sedimentation.

Each molecule floats or sinks to position where density equals density of 

solution (e.g. CsCl gradient for nucleic acid separation). 

Gel Filtration Chromatography

7

Size‐Exclusion Chromatography• Separation of 

proteins based on 

kinetics of moving 

through the available 

space • Larger proteins have 

less space than 

smaller molecules• Proteins larger than 

matrix elute in void 

volume (1 exchange 

of volume outside 

beads)• Proteins smaller than 

matrix partition in 

and out of beads• Pore size in beads is 

not uniform• Also some surface 

interaction with 

beads

Gel Filtration Elution Volumes as a Function of Molecular Weight

Gel Electrophoresis

SDS Gel Electrophoresis

• Tris‐glycine buffer• 10% SDS

Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis

+ electrode

- electrode

Protein Detection in Electrophoresis Gel

• Protein detection– Coomassie blue

– Sypro: green, ruby, Red, orange

– Cybergreen– Silver staining

coomassie brilliant blue A595

Detection of Amino Acids, Peptides, and Proteins

• UV• Biuret Reaction• Ninhydrin Reagent • Fluorescarmine

• Ortho‐phthalaldehyde• Coomassie Brilliant Blue

• Silver Staining

Absorption spectra of Trp & Tyr

Beer’s law: A = εcl. Used to estimate protein concentration

Biuret Reaction

• Primarily measure peptide bonds

• Cupric acid react with the peptide groups of  proteins and form a cupric ion complex 

• Generate purplish‐violet color with Amax

at 540  nm

• Lowry method

• Low sensitivity

Ninhydrin Reaction

• The most widely used reagents for amino acids, peptides, and  proteins

• Reacts with amino groups

• The major step is oxidative deamination of an amino acid to  CO2, NH3, and an aldehyde conatining one less carbon atom  than the original amino acid

• Ninhydrin is simultaneously reduced to ninhydrindantin

• Ninhydrindantin reacts with NH3 and produce purple color  (Amax

570 nm)

• Destroy amino acid or peptide and the material detected  cannot be used for further characterization

Fluorescarmine

• React with amino group

• Produce fluorescent compounds

• Sensitive• The amino group reacted can be recovered by 

hydrolysis and used for further analysis

Ortho‐phthalaldehyde

• React with amino group in the presence of  mercaptoethanol

• Forms a fluorescent reaction product

• Sensitive• As Ninhydrin and fluorescarmin, it is not 

specific for protein

Circular Dichroism Spectroscopy of Polypeptides

Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984

Protein Characterization

• Characterization of proteins and peptides involves  three different processes:

1.

Determining the Amino Acid Composition

• Involves finding out the amino acids that make up the  protein and their number.

2.

Determining the Amino Acid Sequence

• Involves finding out the sequence of amino acids of the  proteins in their order.

3.

Determining the Molecular mass of the Protein

21

• The peptide is first hydrolyzed into its constituent amino acids by heating it in 6M HCl at 110ºC for 24‐72 hrs. 

The R groups remain intact, except for:

• Trp –

indole ring damaged

• Asn, Gln – converted to Asp, Glu

• Gly does not react

22

Determination of Amino Acid Composition

Amino acid analysis

• The amino acids can be derivatized with ninhydrin or o‐phthalaldehyde to make fluorescent derivatives that are 

easy to detect. 

• These are chromatographed by reverse‐phase HPLC (high‐ pressure liquid chromatography). 

– The characteristic retention times are used to identify  the amino acids. 

– The fluorescence level can be quantified to determine  the amount of that amino acid.

Amino acid analysis.

Protein Sequencing Strategy

1) 

Purify protein

2)

Cleave disulfides – react with:A)

reducing agent followed by alkylating agent1)

DTT 

2)

β‐ME

B)

performic acid

Determination of Primary Structure 1

• Hydrolyze peptides with hot 6M HCl.– Identify AA and % of each.– Usually done by chromatography

• Identify the N‐term and C‐term AAs– C‐term via carboxypeptidase

– N‐term via Sanger’s Reagent (DNFB) • Fluoro‐2,4‐dinitrobenzene and dansyl chloride • Phenylisothiocyanate (Edman degradation)

5P2-27

Determination of Primary Structure 2

• Selectively fragment large proteins into  smaller ones.

– Tripsin: cleave to leave Arg or Lys as C‐term AA

– Chymotrypsin: cleave to leave Tyr, Trp or Phe as  C‐term AA

– Cyanogen bromide cleaves at internal Met leaving  Met as C‐term homoserine lactone

5P2-28

Determine AA sequence of peptides

• Edman’s reagent:– First, phenylisothiocyanate reacts with the terminal amino 

group to form a phenylthiocarbamoyl derivative.– This residue cyclizes under acidic conditions to give a 

phenylthiohydantoin (PTH)‐amino acid and a peptide  shortened by one amino acid residue.

– This PTH‐amino acid is identified by HPLC.– The amino acid composition of the shortened peptide can 

be compared with the original peptide.

5P2-29

30

Sanger’s reagent

• Use fluoro‐2,4‐dinitrobenzene and dansyl  chloride

• For N‐terminus amino acid

• Fluoro‐2,4‐dinitrobenzene reacts with an  amino group and forms dinitrophenyl peptide

• Dansyl chloride reacts with dinitrophenyl  peptide and form dansyl peptide

• Acid hydrolysis and analysis using HPLC

Standard Run on 19 PTH AAs

Residue 1 = Leu

Residue 2 = Ile

Protein Sequencing Strategy

• Reassemble sequence through overlaps of  peptides created by different means

• Map disulfides by cleaving protein into peptides  before

disulfide bond cleavage. 

• After purification of disulfide‐linked peptides and  cleavage of their disulfide bonds, sequencing of the 

peptides should reveal which cysteines are linked  in disulfide bonds.

Det. Primary Structure

• A twelve AA peptide was hydrolyzed.• Trypsin hydrolysis: 

– Leu‐Ser‐Tyr‐Gly‐Ile‐Arg

– Thr‐Ala‐Met‐Phe‐Val‐Lys

• Chymotrypsin hydrolysis

–Val‐Lys‐Leu‐Ser‐Tyr

–Gly‐Ile‐Arg

– Thr‐Ala‐Met‐Phe

• Deduce the AA sequence5P2-37

One isC-term

Lys is internal!

Det. Primary Structure

• Tr                                               Leu‐Ser‐Tyr‐Gly‐Ile‐Arg• Ct                                                              

Gly‐Ile‐Arg

• Ct                                 Val‐Lys‐Leu‐Ser‐Tyr• Tr    Thr‐Ala‐Met‐Phe‐Val‐Lys

• Ct   Thr‐Ala‐Met‐Phe

• The complete sequence is:

• Thr‐Ala‐Met‐Phe‐Val‐Lys‐Leu‐Ser‐Tyr‐Gly‐Ile‐Arg

5P2-38

Keeping in mind the N-term AA and overlaping the sequences properly gives:

Protein Identification

• 2D‐GE + MALDI‐MS

– Peptide Mass Fingerprinting (PMF)

• 2D‐GE + MS‐MS

– MS Peptide Sequencing/Fragment Ion Searching

• Multidimensional LC + MS‐MS

– ICAT Methods (isotope labelling)

– MudPIT (Multidimensional Protein Ident. Tech.)

• 1D‐GE + LC + MS‐MS

• De Novo Peptide Sequencing

Isoelectric point (pI)

• Separation by charge:

4

5

6

78

9

10

Stable pH gradient

High pH: protein is negatively charged

Low pH:Protein is positively charged

At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field.

2D‐gel technique example

Advantages vs. Disadvantages

• Good resolution of proteins

• Detection of posttranslational modifications

• Not for hydrophobic proteins

• Limited by pH range • Not easy for low abundant

proteins• Analysis and quantification

are difficult

2D - LC/MS/MS

Study protein complexes without gel electrophoresis

Peptides all bind to cation exchange column

Peptides are separated by hydrophobicity on reverse phase column

Successive elution with increasing salt gradients separates peptides by charge

Complex mixture is simplified prior to MS/MS by 2D LC

(trypsin)

A simple definition of a Mass Spectrometer 

• A Mass Spectrometer is an analytical  instrument that can separate charged  molecules according to their mass–to–charge 

ratio. • The mass spectrometer can answer the 

questions “what is in the sample”

(qualitative  structural information) and “how much is 

present”

(quantitative determination) for a  very wide range of samples at high sensitivity 

Mass Spectrometer

• Works by ionizing molecules and then sorting and  identifying the ions according to their mass‐to‐

charge (m/z) ratios.

• Two key components 

– ion source, which generates the ions– mass analyzer, which sorts the ions

Mass Spectrometer Schematic

Inlet Ion Source

MassFilter Detector Data

System

High Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

Ion Sources

• Electrospray ionization (ESI)

• Atmospheric pressure chemical ionization

(APCI)

• Atmospheric pressure photoionization (APPI)

• Matrix Assisted Laser Desorption Ionization  (MALDI)

High voltage appliedto metal sheath (~4 kV)

Sample Inlet Nozzle(Lower Voltage)

Charged droplets

+++ ++

+

+++ ++

+

+++ ++

+ +++

+++ +++

+++ ++

++

++

+

++++++

+++

MH+

MH3+

MH2+

Pressure = 1 atm Inner tube diam. = 100 um

Sample in solution

N2

N2 gas

Partial vacuum

Electrospray ionization:

Ion Sources make ions from sample molecules (Ions are easier to detect than neutral molecules.)

Desorption of ions from solution

• As the droplets shrink, the charge concentration in thedroplets increases.• The repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. • Useful for analyzing large biomolecules

APCI Ion Source• The LC eluent is sprayed

through a heated (250°C – 400°C) vaporizer at atmospheric pressure.

• The gas-phase solvent molecules are ionized by electrons discharged from a corona needle.

• The solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization).

• The analyte ions pass through a capillary sampling orifice into the mass analyzer.

•

APPI Ion Source

• Avaporizer converts the LC eluent to the gas phase.

• A discharge lamp generates photons in a narrow range of ionization energies.

• The energy ionizes analyte molecules while minimizing the ionization of solvent molecules.

• The resulting ions pass through a capillary sampling orifice into the mass analyzer.

MALDI Ionization

++

+

+

---

++

+

+

----+ +

Analyte

Matrix

Laser

+++

• Absorption of UV radiation by  chromophoric matrix and 

ionization of matrix

• Dissociation of matrix, phase  change to super‐compressed gas, 

charge transfer to analyte  molecule

• Expansion of matrix at supersonic  velocity, analyte trapped in 

expanding matrix plume  (explosion/”popping”)

+

+

+

Mass Analyzers

• Quadrupole• Time‐of‐flight

• Ion trap• Fourier transform‐ion cyclotrone resonance 

(FT‐ICR)

• Mass Spectrometers separate ions according  to their mass‐to‐charge (m/z) ratios 

Quadrupole Mass Analyzer

• Uses a combination of radio frequency and DC

• voltages to operate as a mass filter.

• Has four parallel metal rods.

• Lets one mass pass through at a time. Can scan through all masses or sit at one fixed mass.

Principle of Time of Flight Analyzer

• A uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube.

• Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times.

• Time-of-flight mass analyzers have a wide mass range and can be very accurate in their mass measurements.

Ion Trap 

• Consists of a circular ring  electrode plus two end caps 

that together form a  chamber. 

• Ions entering the chamber  are “trapped”

there by 

electromagnetic fields.• Scanning field can eject ions 

of specific m/z • Advantages 

– MS/MS/MS….. – High sensitivity full scan 

MS/MS 

Fourier transform‐ion cyclotron resonance  (FT‐ICR) Mass Analyzer

• Ions entering a chamber are trapped in circular orbits by powerful electrical and magnetic fields.

• When excited by a radio-frequency (RF) electrical field, the ions generate a time- dependent current.

• This current is converted by Fourier transform into orbital frequencies of the ions which correspond to their mass-to- charge ratios.

• Can perform multiple stages of mass spectrometry without additional mass analyzers.

• Have a wide mass range and excellent mass resolution.

• Very expensive mass analyzers.

What is MS/MS?

MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.

Ion source MS-2MS-1

Mixture of ions

Single ion

Fragments

What is MS/MS?

MS/MS+

+

+ +

+

1 peptide selected for

MS/MS

The masses of all the pieces give an MS/MS

spectrum

Peptidemixture

Have only masses to start

Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle

+

+

+ +

+

Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together

Tandem Mass Spectrometry

• Different MS‐MS configurations

– Quadrupole‐quadrupole (low energy)– Magnetic sector‐quadrupole (high energy)

– Quadrupole‐time‐of‐flight (low energy)

– Time‐of‐flight‐time‐of‐flight (low energy)

• Fragmentation experiments may also be performed on  single analyzer instruments such as ion trap instruments 

and TOF instruments equipped with post‐source decay

Tandem MS

Fragmentation in MS/MS produces sequence‐specific  fragment ions

Mass Spectra

• The peaks in the mass spectrum:– Prefix – Fragments with neutral losses

(‐H2

O, ‐NH3

)– Noise and missing peaks.

G V D L K

mas s0

57 Da = ‘G’ 99 Da = ‘V’LK D V G

and Suffix Fragments.

D

H2O

Protein Identification with MS/MS

G V D L K

mas s0

Inte

nsity

mas s0

MS/MSPeptide Identification:

Peptide sequencing by MS/MSPeptide sequencing by MS/MS

(Standing 2003 Curr. Opin. Struct. Biol. 13, 595-601)

Protein Identification by Tandem Mass  Spectrometry

SSeeqquueennccee

S#: 1708 RT: 54.47 AV: 1 NL: 5.27E6T: + c d Full ms2 638.00 [ 165.00 - 1925.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

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95

100

Rel

ativ

e A

bund

ance

850.3

687.3

588.1

851.4425.0

949.4

326.0524.9

589.2

1048.6397.1226.91049.6489.1

629.0

MS/MS instrumentMS/MS instrument

Database search•Sequestde Novo interpretation•Sherenga

Advantages vs. Disadvantages

• Determination of MW and aa. Sequence

• Detection of posttranslational modifications

• High-throughput capability

• High capital costs• Requires sequence

databases for analysis