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
Joaquín Abián 2002, EMEB IIBB-CSIC
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.
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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.
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
Joaquín Abián 2002, EMEB IIBB-CSIC
HUMAN PLASMA
Joaquín Abián 2002, EMEB IIBB-CSIC
CÉLULAS•HT93 (Tirocitos)•Astrocitos•PKC (riñón)•PC12 (hígado)
TEJIDO•Hígado•Riñón•Tirocitos humano•Embrión (Maíz)
COMPARTIMENTOS•Mitocondria
MICROORGANISMOS•E. coli
Joaquín Abián 2002, EMEB IIBB-CSIC
CÉLULAS•HT93 (Tirocitos)•Astrocitos•PKC (riñón)•PC12 (hígado)
TEJIDO•Hígado•Riñón•Tirocitos humano•Embrión (Maíz)
COMPARTIMENTOS•Mitocondria
MICROORGANISMOS•E. coli
Joaquín Abián 2002, EMEB IIBB-CSIC
CÉLULAS•HT93 (Tirocitos)•Astrocitos•PKC (riñón)•PC12 (hígado)
TEJIDO•Hígado•Riñón•Tirocitos•Embrión (Maíz)
COMPARTIMENTOS•Mitocondria
MICROORGANISMOS•E. coli
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
Search for protein markers of renal injurythat could by used as drugs targets or diagnostic markers
in the treatment of acute renal failure
OBJECTIVES
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
CONTROL ISQUEMIA NONOs + ISQUEMIA
PKCs DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS
12
Joaquín Abián 2002, EMEB IIBB-CSIC
Hereditary cardiomyopathy
Normal
Disease(underexpression)
Normal
Disease(overexpression)
Weekes et al, Electrophoresis 1999, 20, 898
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
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
Joaquín Abián 2002, EMEB IIBB-CSIC
Silver stained 2-D gel electrophoresis separation ofproteins from transfected Human Np18 cells
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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)
Joaquín Abián 2002, EMEB IIBB-CSIC
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)
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
PROTEIN IDENTIFICATION
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
Ion trap-MSwith
nanoESI source
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
PEPTIDE MAPPING
PEPTIDE MASS LIST
PEPTIDE Mass
1 345.22 678.33 768.24 1005.35 1252.26 1380.2
PROTEOLISIS
MASS ANALYSIS
DATABANKSEARCH
MATCH !
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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+
Joaquín Abián 2002, EMEB IIBB-CSIC
HUMAN PLASMA
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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?
Joaquín Abián 2002, EMEB IIBB-CSIC
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?
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
• 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
Joaquín Abián 2002, EMEB IIBB-CSIC
Minor Protein Enrichment
Chromatography
Subcellular fractionationMetal Affinity chromatography
2-D Chromatography
ImmunopurificationAffinity chromatography
Labelling+Digestion+AffinityMethods
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
WHY MINIATURIZED LC-ESIMS SYSTEMS?
Better recovery for peptides at low levels High sensitivity Electrospray techniques work at low flow rates
Joaquín Abián 2002, EMEB IIBB-CSIC
COLUMNAS AUTOCONSTRUIDAS PARA CROMATOGRAFÍA CAPILAR
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
PROTEIN IDENTIFICACION USING MASS PROTEOMICS
PEPTIDE MASS MAPPING
GEL ELECTROPHORESIS
IN GEL DIGESTION
DESALTING (µLC)
NANOELECTROSPRAY(MS/MS, MSn)
MATCH !
PROTEIN IDENTIFICATION
MALDIOn-line µLC-
µELECTROSPRAY(MS/MS)
MS & MS/MS #5MS & MS/MS #4
MS & MS/MS #3MS & MS/MS #2
MS & MS/MS #1
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
PULLED CAPILLARY COLUMN AND INTERFASE
Joaquín Abián 2002, EMEB IIBB-CSIC
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
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
Joaquín Abián 2002, EMEB IIBB-CSIC
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
Joaquín Abián 2002, EMEB IIBB-CSIC
ProteomicsHOT R&D AREAS
Postraslational Modifications
STRUCTURE, FUNCTION
®ULATION
Protein interactions
R&DSeparative Techs
R&DMass Spec Meths
Profiling Quantitation
Protein arrays
Target discoveryDrug&Target discovery
Joaquín Abián 2002, EMEB IIBB-CSIC
Prot complexes
Protein interactions
Prot/Prot & otherinteractions
Epitope mapping
ProteomicsHOT R&D AREAS
Therapy, drug development
Cell functions & structureMetabolic PathwaysMechanism of action
Mol Biol Protein ChemBiochemistry
Genomics Protein structureCell Biology
Joaquín Abián 2002, EMEB IIBB-CSIC
Postraslational Modifications
Splicing/Metab.
Phosphorylation Glycosylation
ProteomicsHOT R&D AREAS