Definiciones • Una visión global? • Estrategia...

Joaquín Abián 2002, EMEB IIBB-CSIC

INTRODUCCION A LA PROTEOMICAINTRODUCCION A LA PROTEOMICA

• Definiciones• Una visión global?• Estrategia analítica• Cantidad y variedad• Más allá de la identificación

J.AbianBiological and Structural Mass Spectrometry Unit

Medical Bioanalysis, IIBB/CSIC-IDIBAPS, Barcelona, Spain


1The protein equivalent of a genome

M.Williams, 1994 Sienna Meeting

PROTEOMEProte(in)-(Chromos)ome

2The protein pattern of an organism, a cell, an organelle or a body fluid

determined quantitatively at a certain momentand under precisely defined conditions

F.Lottspeich, 1999 Angew.Chem.Int.Ed.


Genoma

Transcriptoma

Proteoma

MetabolomaHigh density cDNA arrays are poor

predictors of intracellular protein abundance

Once synthetized, mRNA moleculesare destroyed by endonucleases

at different rates

mRNA half lives vary from minutes to hours

Protein function is controlledby postraslational modifications

Final amounts of funtional protein aredependent on maturation and degradation processes

The amount of mRNA doesnt allows conclusionson the amount of synthetized active protein


PROTEOMICAPROTEOMICA

La proteómica estudia los cambios cuantitativos en un determinado proteoma y su aplicación en diagnóstico de enfermedades, terapia y desarrollo de fármacos.

Las herramientas básicas de la proteómica actual se asientan en dos técnicas principales:la electroforesis en gel como método de separación y selección de proteínas diana

la espectrometría de masas como método de identificación y secuenciación.

Estas técnicas se benefician de los conocimientos ya existentes sobre el genoma y proteoma humano y de otras especies. Esta información se encuentra disponible en bases de datos de libre acceso y puede utilizarse mediante programas de búsqueda específicos

que relacionan las secuencias en la base de datos con los datos espectrométricos obtenidos.


95% of drug targets are proteins

> 500.000 proteins in human proteome (~ 40.000 genes) 95% are largely uncharacterized, and functions cannot be assigned

Current drug targets ~ 500

PROTEOMICS AND DRUG DISCOVERYPROTEOMICS AND DRUG DISCOVERY

Potential Proteomic drug Targets ~ 10.000


PROTEOMIC MARKET

Global Growth

$565 millions (2001) $3300 millions (2006) +40% annual

Proteomic Services+50% annual

Protein Chip Segment

$65.7 millions (2001) $723 millions (2006) +62% annual

$311 millions (2001) $1700 millions (2006)

Proteomic Platforms


MASS PROTEOMICSMASS PROTEOMICS

TWO PROTEOMES ARE COMPARED AND DIFFERENCES STUDIEDDo not requires previous knowledge of the biological system

2-D gels are needed to resolve the components.Observed differences are often not directly related to the problem

SUBSETS OF RELATED PROTEINS ARE STUDIEDSubsets obtained by (for example) affinity separation procedures

Components can be resolved in 1-D gelsMore effective in solving problems

CLASSICAL PROTEOMICS

FUNCTIONAL (CELL MAP) PROTEOMICS


HUMAN PLASMA


CÉLULAS•HT93 (Tirocitos)•Astrocitos•PKC (riñón)•PC12 (hígado)

TEJIDO•Hígado•Riñón•Tirocitos humano•Embrión (Maíz)

COMPARTIMENTOS•Mitocondria

MICROORGANISMOS•E. coli


CÉLULAS•HT93 (Tirocitos)•Astrocitos•PKC (riñón)•PC12 (hígado)

TEJIDO•Hígado•Riñón•Tirocitos•Embrión (Maíz)

COMPARTIMENTOS•Mitocondria

MICROORGANISMOS•E. coli


PKCs DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS

106 Cells

Control Isquemia NONOs + Isquemia

7h in hipoxic chamber0.5% O2 + 19% CO2

7h in hipoxic chamber0.5% O2 + 19% CO2

NONOs 500 µM

Hotter & Abian 2003


Search for protein markers of renal injurythat could by used as drugs targets or diagnostic markers

in the treatment of acute renal failure

OBJECTIVES


PIG KIDNEY CELLS (PKCs)

A

Apa

rent

Mol

ecul

ar w

eigh

t (K

Da)

Isoelectric Point4 5 6 7

37

25

50

75

15

ControlB

Apa

rent

Mol

ecul

ar w

eigh

t (K

Da)

Isoelectric Point4 5 6 7

37

25

50

75

15

Isquemia

12


CONTROL ISQUEMIA NONOs + ISQUEMIA

PKCs DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS

12


Hereditary cardiomyopathy

Normal

Disease(underexpression)

Normal

Disease(overexpression)

Weekes et al, Electrophoresis 1999, 20, 898


Study of Protein expression in sera from TOS patientsStudy of Protein expression in sera from TOS patients

Genomic studies on the xenobiotic enzyme metabolism in TOS survivors suggested that individual metabolic factors could play an importan role in the occurrence of the disease.

It has been demonstrated that NAT2 activity is significativelly reduced in the TOS-affected population.

Differential protein expression analysis in serum

Search for protein markers of disease or disease predisposition

OBJECTIVES

BACKGROUND


Differential protein expression analysis in human serum

C.Quero et al., UCO2003

CONTROL TOS PATIENT

49

35

29

Isoelectric PointEl

ectro

phor

etic

mob

ility

(KD

a)4 5 6

49

35

29

4 5 6Isoelectric Point


TTR

s

a2

TTR

s

Hp 1s-1s

TTR

f

Hp 1f-1f Hp 1s-1f

TTR

f

a2

Hp 2-1f Hp 2-1s

TTR

a2

Hp 2-2

TTR

s f

Haptoglobin alpha Phenotypes


CONTROL CASE

MALEage 11-20

MALEage 21-30

MALEage 31-40

FEMALEage 51-56

P07 P16

P02 P04

P13 P23

P11 P20

TTR

f

a2

Hp 2-1f

TTR

a2

Hp 2-2

TTR

s

Hp 1s-1s

Averaged phenotypes of sera pools

TTR

s

Hp 2-1sa2

Study of Protein expression in sera from TOS patientsStudy of Protein expression in sera from TOS patients

Haptoglobin phenotype can differentiate between control and TOS suffering individuals.

C.Quero et al., Proteomics (2003) In press


1Set of proteins whose alteration in expression is characteristic of

a response to a defined condition or genetic changeNeidhardt, F.C. and co-workers, Electrophoresis, 1999

PROTEOMIC SIGNATURE

Joaquín Abián 2002, EMEB IIBB-CSICMiller et al, Electrophoresis (1999), 20, 836

Rat serum

5 µl sampleCoomassie staining

Effect of turpentine

Control maleInflamed maleControl femaleInflamed female

2-D Gel Analysis


1Set of proteins whose amount of synthesis rate

changes in response to a single stimulusNeidhardt, F.C. et al 1990

STIMULON

1Set of proteins whose synthesis is regulated by the same regulatory protein

Neidhardt, F.C. et al 1990

REGULON


Silver stained 2-D gel electrophoresis separation ofproteins from transfected Human Np18 cells


Proteomics: A Global View ?How many proteins are out of the firing range?

H. Kaji et al., Electrophoresis 2000, 21, 1755

C.elegans database,Predicted IP and Mr (19126 proteins)

Only about 11000 spotsin conventional

2D gel electrophoresis range


Proteomics: A Global View ?Which resolving power do we have?

Silver stained 2-D gel electrophoresis separation ofproteins from transfected Human Np18 cells

Resolving power: 11000 spots(7% of the human proteome)

A high number of spotscontain two or more proteins

20 % procariotes (Link, Electrophoresis 1997)40% eucariotes (Parker, Electrophoresis 1998)


Análisis de expresión diferencial en hígado de rata

49

35

29

49

35

29

Punto isoeléctricoA B4 5 6

Peso

mol

ecul

ar a

pare

nte

(KD

a)Punto isoeléctrico

4 5 6

CONTROL PANCREATITIS

disulfuro isomerasa ER-60(J. Abian & D. Closa, en preparación)


PROTEOMICS: ANALYTICAL STRATEGY

SAMPLE 1 SAMPLE 2

SAMPLE PREPARATION

(SAMPLE FRACTIONATION)

2-D GEL ELECTROPHORESIS

IMAGING & SUBSTRACTIVE ANALYSIS

PROTEIN IDENTIFICATION

No PCR type technology for proteins

Proteins analysis is thus restricted by1) protein levels in the sample and

2) detection limits of the analytical system

Proteome is more complexthan transcriptome

Several protein species can be producedfrom each gene

(The protein tau shown more than 100 spots by 2D in human brain)

Protein levels are very different

Abundance ratios as high as 10E7


In gel digestion(trypsin, V8, endolysine C, Arg-C)

Mass analysisPeptide Mass Mapping (MALDI-TOFMS)

Peptide sequencing (ESI-MS/MS)

Database Mining(SwissProt, NCBInr, Genbank, dbEST)

Database Mining(Protein Prospector, MASCOT, SEQUEST)

1ª Separation by isoelectric point(7-24 cm immobiline strips pI 3-10)

(zoom strips 1 pI unit range)

2ª por mobilidad electroforética (“masa”)(homogeneous and gradient gels)

Visualizacion de las proteinas(Plata, Coomassie, Zinc-Imidazol,

SyPro Ruby, radiactivity detection)

Tissue homogeneization(Polytron, Sonication)

Cell Lysis and protein solubilization(Urea 8 M, Chaps, leupeptin, DTT, TRIS)

Solubilización del mayor número de proteínasEtapa crítica

Biological tissue or fluidIsolated or culture cells

Cellular organelle(Maybe prelabelled with 35S-Met/Cys, 32P-ATP)

PROTEOMICS: ANALYTICAL STRATEGY

SAMPLE 1 SAMPLE 2

SAMPLE PREPARATION

(SAMPLE FRACTIONATION)

2-D GEL ELECTROPHORESIS

IMAGING & SUBSTRACTIVE ANALYSIS


ImmunoprecipitationAffinity chromatography

(Ab, heparin, protein, Oligo)Metal Affinity Chromatography (IMAC)

Size exclusion chromatographyLocalización de spots

Quantificación y Análisis diferencialAnálisis de componentes principales

Análisis de clustersIdentificación de spots de interés


SECTOR

TIME OF FLIGHT

QUADRUPOLE

ION TRAP

ION SOURCE ANALYZER ION DETECTOR

¿What?Precise determination on molecular masses of molecules and aggregates

Breakdown of molecules in fragments

¿How?Production of gas phase ions from the molecule

Determination of the mass to charge ratio by interactionwith electric, magnetic or radiofrecuency fields

¿Why?Confirmation of chemical structure

Identification of unknown compounds in mixturesCaracterization of known molecules

Determinación of peptide and proteins sequenceQuantification

Study of non-covalent interactionsDrug-receptor interaction

Study of Protein conformation

ESPECTROMETRIA DE MASAS


Electrospray

Fast Atom Bombardment

Matrix Assisted Laser Desortion

3 kV

252Cf Plasma Desorption

¼ Nobel Prize 2002

Koichi Tanaka

¼ Nobel Prize 2002

John Fenn


MALDI-TOFMS

800 1440 2080 2720 3360 4000

20

40

60

80

100

DR4DM (Fracción 33)

852.41

2152.06

1529.82

1141.592769.15

2192.17877.03 1797.86892.99 2759.551329.57 1608.781297.74

Determinación de pesos moleculares

2128 2146 2165 2183 2201

Mass (m/z)

2152.056

2151.054

2153.063

2192.171

2154.066 2191.1912193.180

Determinación de pesos moleculares(Con alta resolución y precisión)

0

20

40

60

80

100

% In

tens

ity


ESPECTROMETRIA DE MASASBIOLOGICA

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

1344.3

1245.41116.4

928.3

772.2

1444.4949.1 1048.2

1606.41177.2625.21522.3850.1

1735.5708.2 1668.3

524.11806.5

1864.6431.0 1934.8

y14

y15

y16 y17 y18

y9

y10y11

y13

y4

y5

y6

y12

1030.4

WGDAGAEYVVESTGVFTTmEKMr 2292 Da

y8

b17

b11b10

b9

b8 b16

b13b12b4

MS/MS spectrumion 1147

S#: 1 RT: 0.04 AV: 1 SM: 7B NL: 9.74E6T: + p Full ms

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1060.5998.1942.8

1131.0

893.31211.7

848.7

1304.8

808.2

1413.3524.3 771.6

1541.9738.01309.31214.3707.3 1695.9

1546.7694.3

MYOGLOBIN (HORSE) nanoESI

40 fmol total amountMr = 16950

RANGO DE MASA>30 KDa - >200KDa

PRECISION>0.05% - 0.008%

LIMITES DE DETECCIONfmol-attomol

RESOLUCION (M/∆M)>500 - >3500

ESTRUCTURACID, PSD

CARACTERISTICAS


Ion trap-MSwith

nanoESI source


MATCH !

HUELLA PEPTIDICAPéptido Masa

1 345.22 678.33 768.24 1005.35 1252.2

MALDI-MS

ESI-MS/MS

400 600 800m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce 840.2

727.1709.2

569.2626.2 822.3456.1

385.1

MGL(I)A T

Células, tejido, fluido

control enfermedad

Immunopurificación Complejosproteicos

digestiónPieza de gel

Electroforesis 2D

SDS-PAGE

Buscador

Basede datos

Huella de secuencia

PROTEOMICA: ESTRATEGIA ANALITICA


PEPTIDE MAPPING

PEPTIDE MASS LIST

PEPTIDE Mass

1 345.22 678.33 768.24 1005.35 1252.26 1380.2

PROTEOLISIS

MASS ANALYSIS

DATABANKSEARCH

[email protected]

MATCH !


PEPTIDE MAPPING

749.0 1499.2 2249.4 2999.6 3749.8 4500.0Mass (m/z)

8783.2

20

40

60

80

100

% In

tens

ity

1433.672

1493.737842.510 2963.4771964.952 2677.230

2781.40071994.982 3325.7151045.419 2240.233


Accuracy of mass measurements and protein identification

C.elegans97 Mbase genome19100 predicted proteins918655 possible tryptic peptides

Average Accuracy

MALDI-QUAD 100 ppmMALDI-TOF 5-30 ppmMALDI-FTICR 1 ppm

R.D.Smith et al, Electrophoresis 2001, 22, 1652


A -- B -- C -- D -- E100 125 90 110 175

D - EC - D - E

B - C - D - EA - B - C - D - E

E

Mr = 600 DaMr = 500 DaMr = 375 DaMr = 285 DaMr = 175 Da

PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY

mass/charge

010

2030

40506070

8090

100

200 400 600300 500100

Peptide ABCDE600

mass/charge

010

2030

40506070

8090

100

200 400 600300 500100

E D C B A

600

500375175

285


WGDAGAEYVVESTGVFTTmEKMr 2292.0 Da

MS/MSm/z 1147.0

400 600 800 1000 1200 1400 1600 1800 2000

20

40

60

80

100

m/z

Rel

ativ

e Ab

unda

nce

b10+

y14+

y15+

y16+

y17+

y18+

y9+

y10 + y11

+

y13+

y4+

y5+

y6 +

y12 +

y8 +

b16+

b11+

b9+

b8+ b15

+

b13+

b4+

T F V G T S E V V Y E A G A

1344.3

1245.41116.4

928.3

772.2

1444.4949.1 1048.2

1606.41177.2625.2

1522.3850.11735.5708.2 1668.3

524.11806.5

1864.6431.0 1934.8

1030.4

A G A E Y V V E S T G V F T T

b5+ b6

+b7

+

b17+

b18+

b3+

y7+


HUMAN PLASMA


Procedure

1. Fix gel in 40% EtOH, 10% HAc for 1 hr.

2. Wash gel in 30% EtOH, 2 x 20 mins.

3. Wash gel in H2O for 20 mins.

4. Sensitize gel in 0.02% Na2S2O 3 for 1 min.

5. Wash gel in H2O, 3 x 20 secs.

6. Incubate gel in cold 0.1% AgNO3 , 20 mins. at 4ºC

7. Wash gel in H2O, 3 x 20 secs.

8. Change gel chamber

9. Wash the gel in H2O for 1 min.

10. Develop gel in 3% Na2CO3 , 0.05% formalin

Observe the color and change solution when the developer turns yellow. Terminate when the staining is

sufficient.

11. Wash the gel in H2O for 20 secs.

12. Terminate staining in 5% HAc

13. Wash the gel in H2O, 3 x 10 mins.

14. Leave the gel at 4ºC in 1% HAc

Blum Silver Staining Protocol (modified)

Proteomics: A Global View ?How much protein do we need?


Peptide recovery determined as BSA sequence coverage by MALDI peptide maps.

A. Görg and cols. ASMS

(1) Rosenfeld(2) Shevchenko et al. Anal.Chem. 68, 850-858, 1996.(3) Blum et al. Electrophoresis, 8, 93-99, 1987.(4) Vorum et al. Exp. Cell Res., 248, 473-481, 1999.(5) Gottlieb et al. Anal.Biochem.165, 33, 1987.(6) Gharahdaghi et al. Electrophoresis, 20, 601-605,1999.

1 pmol

500 fmol

100 fmol

50 fmol

25 fmol

10 fmol

5 fmol

Comassie (1) 35% 40% 23% - - - - EMBL (2) 64% 59% 48% 31% 16% 10% 8% Blum (3) 57% 59% 44% 26% 26% 12% 7% Vorum (4) 57% 50% 37% 23% 21% 19% - BioRad (5) 44% 45% 19% - - - - Destaining (6) 46% 36% 22% 11% 11% - -

Effect of staining methodHow much protein do we need?


I. Lefkovits et al., Electrophoresis 2000, 21, 2688

Lymphoblast (stimates)

Proteomics: A Global View ?How many proteins are we actually seeing?

M. Perrot et al., Electrophoresis 1999, 20, 2280

Yeast (S288C)

100 diff proteins

1100 diff proteins

2800 diff proteins


1) 5000 PROTEINS IN THE MIXTURE (YIELDING 40,000 TRYPTIC FRAGMENTS)

2) AVERAGE MOL. WT. OF PROTEINS = 30 KDa

3) YOU NEED AT LEAST 15 FMOL TO GET A PEPTIDE SEQUENCE VIA MS/MS

(450 pg OF EACH PROTEIN) x (5000 PROTEINS) = 2.25 µg TOTAL PROTEIN

Andrew J. Alpert, Ph.D.PolyLC Inc.

HOWEVER, PROTEINS DIFFER BY 10 6 IN REL. ABUNDANCE. RELATIVELY FEWPROTEINS MAKE UP MOST OF THE SAMPLE.

TO GET 15 FMOL OF THE LEAST ABUNDANT PROTEIN, YOU WILL NEED AT LEAST 10 5 MORE SAMPLE:

2.25 µg x 10 5 = 225 mg TOTAL PROTEIN NEEDED

SAMPLE WEIGHT AND PROTEIN LEVELS


Low copyLow copy(10.000)(10.000)

MediumMedium(60.000)(60.000)

High copyHigh copy(9 M)(9 M)

TOTALTOTAL

15 fmols15 fmols0.1 pmols0.1 pmols15 pmols15 pmolsProtein per spotProtein per spot(10(1066 cells, 670 ug)cells, 670 ug)

2.22.24.84.860606767Weight (pg per cell)Weight (pg per cell)

280028001100110010010040004000Number of protein Number of protein speciesspecies

PROTEIN ABUNDANCEPROTEIN ABUNDANCELymphoblast cell: 670 pg10% (67 pg) is protein

I. Lefkovits et al., Electrophoresis 2000, 21, 2688

SAMPLE WEIGHT AND PROTEIN LEVELS

2-D Gel Loadability50 -300 µg

0.5-1 million cells


• Low copy proteins (<100.000 copies per cell)•Trasiently modified proteins (phosphorylated...)

Minor Proteins

Codon bias studies suggest more than 50%coded proteins still remain below routine detection limits

(codon bias<0.1) (transcription factors, protein quinases)

Protein Enrichment


Minor Protein Enrichment

Chromatography

Subcellular fractionationMetal Affinity chromatography

2-D Chromatography

ImmunopurificationAffinity chromatography

Labelling+Digestion+AffinityMethods


Profiling

Universal Visualization

Family Profiling

Extreme pK proteinsHydrofobic ProteinsCell membrane and walls

Microbial pathogenicity/drug resistance determinants

Cause-Effect Profiling(expression proteomics)

RegulonsStimulons

Proteomic Signature

Cellular state prot. markersDiagnostic & Cell-Aggression markers

ProteomicsHOT R&D AREAS

Bioinformatics

Minor proteins Low MW proteins


ProteomeDigest 2Digest 1

Digest 3

Digest n

Protein 3

Protein 1Protein 2

Protein n

Digest 1Peptide 2Peptide 1

Peptide 3---------Peptide n

---------

Fingerprintingand/or

Peptide Sequencing

AutomatedLC(/LC)-MS/MS

Separation+Identification (1 analysis)

Conventional vs Shotgun Proteomic Analysis

Digest

Separation (n analyses)Digest Identification

Prot AProt BProt C

Prot Z----------------

Prot AProt BProt C

Prot Z-------

Proteome

Coupling Liquid phase separative techniques with Mass Spectrometric detection

Protein DigestSubproteome digestProteome digestProtein mixture

HPLCCECCZE

COMPONENT SEPARATION MASS ANALYSIS

MS

Total ion chromatogramV

elution time

Separate chemical species for individual analysis- Additional dimension two the MS análysis- Eliminates ionization or mass interferences

Reproducible

Automatable

mass/charge

spectrum 234 (elution time 3.5 min)

V


MS capillary inletMS INTERFACE

SS Ferrule EXC

Teflon sleeve

FSC

Capillary columnInjectorHPLC pump

Flow splitter

T

M.Carrascal and J.Abián in HPLC of Peptides and Proteins, Methods in Molecular Biology Series (2003) In Press

The micro/nanoLC-µESIMS setup


WHY MINIATURIZED LC-ESIMS SYSTEMS?

Better recovery for peptides at low levels High sensitivity Electrospray techniques work at low flow rates


COLUMNAS AUTOCONSTRUIDAS PARA CROMATOGRAFÍA CAPILAR


mAU

0

50

100

150

200

250

30013

.514

.214

.8

15.8

17.2

YGGFMK

DRVYIHPFHL

VEPIPY

QATVGDINTERPGMLDFTGKDRVYIHFHLLVYS

SPGFR

Kromasil C8, 90 x 0.250 mm6-35 pmols

4 µl/minmAU

0

50

100

150

200

250

300

15.4

16.2 16

.8 17.4

18.3

YGGFMK

DRVYIHPFHL

QATVGDINTERPGMLDFTGKDRVYIHFHLLVYS

SPGFR

VEPIPY

Kromasil C18, 100 x 0.250 mm6-35 pmols

4 µl/min

MICRO-LIQUID CHROMATOGRAPHY OF PEPTIDES

min8 12 1610 18 2014 min8 12 1610 18 2014


NanoLC-µESI-MS

0

Time (min)10 20 30

Rel

ativ

e A

bund

ance

VHLTPVEK

TGGFM

VEPIPY

QATVGDIDTERPGMLDFTGK

Angiotensinogen (1-14)

2,11 Ala-ET

5 µm Kromasil C18

75 µm i.d. X 10 cm(190 µm o.d.)

H2O/ACN/MeOHgradient1%AcOH

0.05% TFA

550 nL/min2.5 µL inject

75-400 fmols on column

0

25

50

75

100

A.Oosterkamp et al, J. Mass Spectrom. 33, 976 (1998)

Peptide mapping by µLC-ESIMS

RT:19.85 - 70.07

35Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

43.18

52.58

48.74

42.5640.78

43.57

38.72

46.4336.57 54.05

33.74 54.8231.11

20

30 40 45 50 55 60 65 702554.0 55.0 56.0 57.0 58.0 59.0

Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

56.4

55.1

56.6

56.7

55.0 56.856.3

55.357.756.9

58.056.1 57.6

Finnigan LCQDependent scan mode

MS300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

858.3

840.4456.2 569.3 727.4

MS SCAN AT 57.6 min

300 400 500 600 700 800 900m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

840.2

727.1709.2

569.2

626.2 822.3456.1

385.1

MS/MS SCAN AT 57.7 minPRECURSOR 858.3

MGL(I)A T

MS/MS

AUTOMATED MS/MS SEARCH


PROTEIN IDENTIFICACION USING MASS PROTEOMICS

PEPTIDE MASS MAPPING

GEL ELECTROPHORESIS

IN GEL DIGESTION

DESALTING (µLC)

NANOELECTROSPRAY(MS/MS, MSn)

MATCH !


MALDIOn-line µLC-

µELECTROSPRAY(MS/MS)

MS & MS/MS #5MS & MS/MS #4

MS & MS/MS #3MS & MS/MS #2

MS & MS/MS #1


CHARACTERIZATION OF THE 98kDa GLYCOPROTEIN rBAT FROM A SILVER STAINED GEL

lc Ab

P4N1 b0,+ AT

hc Ab

36.3 -

94 -

b0,+AT

???

kDa

¿ Se asocia rBAT con b0,+AT “in vivo”?

- Genes asociados a cistinuria:. SLC3A1: Codifica por la proteina rBAT. SLC7A9: Codifica por la proteina b0,+AT

- Cistinuria: Enfermedad congénita debida a un defecto en el transporte de aminoácidos

Inmunoprecipitación de b0,+AT de riñón de ratón

SDS-PAGE Tinción con plata

E.Fernández, M.Carrascal et al. AJP-Renal Physiology, on-line March 19, 2002


PULLED CAPILLARY COLUMN AND INTERFASE


LCQ ion traptriple play + dynamic exclusion

RT: 20.00 - 85.00

20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

51.69

59.75

49.93

46.3752.67

30.25

46.69 55.5235.57 37.9630.85 63.4927.61 43.13 75.1079.21

38.79 65.56 72.44 79.8468.8122.41 23.99

Characterization of the 98kDa Glycoprotein#MP01 from a silver stained gel

EVLFQFSGQAR

Optimized µLC-µESIMSinterface

Identified protein Specie Seq. peptides

NBAT M musculus 41

Meprin A alpha subunit M musculus 1

Ig mu heavy chain from prepeptide

O. cuniculus 3

Trypsin B. taururs 6

Keratin H.sapiens 7

Non identified spectra - 5


1 11 21 31 41 51 61 71MDEDKGKRDP IQMSLKGCRT NNGFVQNEDI PEQDPDPGSR DTPQPNAVSI PAPEEPHLKA VRPYAGMPKE VLFQFSGQAR81 91 101 111 121 131 141 151YRVPREILFW LTVVSVFLLI GATIAIIVIS PKCLDWWQAG PIYQIYPRSF KDSDKDGNGD LKGIQEKLDY ITALNIKTLW 161 181 191 201 211 221 231ITSFYKSSLK EIDPFFGTMK DFENLVAAIH DKGLKLIIDF IPNHTSDKHP WFQSSRTRSG KYTDYYIWHN241 251 261 291 301 311CTHVNGVTTP PNNWLSVYGN SSWHFDEVRK VQEEIKEIIT FWLSKGVDGF SFDAVKFLLE 321 331 341 351 361 371 381 391AKDLRNEIQV NTSQIPDTVT HYSELYHDFT TTQVGMHDIV RDFRQTMNQY SREPGRYRFM GAEASAESIE RTMMYYGLPF401 411 421 431 441 451 461 471IQEADFPFNK YFTTIGTLSG HTVYEVITSW MENMPEGKWP NWMTGGPETP RLTSRVGSEY VNAMHMLLFT LPGTPITYYG481 491 501 511 521 531 541EEIGMGDISV TNFNESYDST TLVSKSPMQW DNSSNAGFTE ANHTWLPTNS DYHTVNVDVQ KTQPSSALRL 561 571 581 591 601 611 621 631ELVLSRGWFC LLRDDSHSVV YTRELDGIDN VFLVVLNFGE SSTVLNLQGI ISDLPPELRI RLSTNSASKG SAVDTRAISL641 651 661 671 681EKGEGLVLEH STKAPLHQQA AFRDRCFVSS RACYSSALDI LYSSC

171DFRYAVEDFK

271QCYFHQFLKE

281QPDLNFRNPA

551YQDLSLLHAT

Red aa sequenced by capillary LC-MS/MS.

Yellow aa differ from the database sequence.

Characterization of the 98kDa Glycoprotein #MP01from a silver stained gel

- Protein : 685 aa-Sequenced : 261 aa ( 41 tryptic peptides)-Coverage: 38%

DFRYAVED F K

F179 was anotated asI (I →→→→ F)

in Genpept and Tremb

QCYFHQFL K E

K279 was anotated asR (R →→→→ K)

in NCBI database

QPDL N FRNPA

N285 was anotated asY (Y →→→→ N)

in NCBI database

YQDLSLL H AT

H558 was found bothas histidine

and as oxo-histidine



Postraslational Modifications

STRUCTURE, FUNCTION

&REGULATION

Protein interactions

R&DSeparative Techs

R&DMass Spec Meths

Profiling Quantitation

Protein arrays

Target discoveryDrug&Target discovery


Prot complexes

Protein interactions

Prot/Prot & otherinteractions

Epitope mapping


Therapy, drug development

Cell functions & structureMetabolic PathwaysMechanism of action

Mol Biol Protein ChemBiochemistry

Genomics Protein structureCell Biology


Postraslational Modifications

Splicing/Metab.

Phosphorylation Glycosylation


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