Proteomics of body liquids as a source for potential methods for medical diagnostics

and mass spectrometry

Prof. Dr. Evgeny Nikolaev

Institute for Energy Problems of Chemical Physics and Institute for biochemical physicsRus. Acad. Sci., Moscow, Russia.

Modern biological mass spectrometers are mainly

ESI- TOF Measuring time of ion flights in vacuumMALDI-TOF

Orbitraps Measuring frequencies of ion oscillations

Ion traps Measuring ion motion stability parameters

FT ICR Measuring frequencies of ion oscillations in magnetic field

Ortho – ESI TOFBruker, Thermo, Applied Biosystems, Agilent, Waters…….

OrthogonalTOF Dodonov 1986

Reflectron Mamyrin 1973

Electrospray Gall 1984

API Ion source Linear Ion Trap C-Trap

Orbitrap

differential pumping

differential pumping

The Thermo Scientific* LTQ Orbitrap XL* hybrid FTMS

Alexander Makarov Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal. Chem. 2000;72: 1156.

The main goal of our research is to connect the level of protein expression with diseases or to find disease biomarkers.

Our Project:

Protein

enzym Mass analysesfragmentation

Isolated peptide

Masses of peptide fragments

Search in database

scoringProtein and DND sequence database

Mass analyses

High throughput proteome analyses by

tandem mass spectrometry methodsBottom-up method

Protein

энзим анализ масс 1

Массы фрагментовпептидов

Поиск в базе

Tор-down method - direct mass spectrometry of proteins and peptides

Ion transportation

Mass analyses

Masses of peptide fragments

Search in database

scoringProtein and DND sequence database

fragmentation

KETAAAKFERQYL

K ETAAAKFERQYLKE TAAAKFERQYLKET AAAKFERQYLKETA AAKFERQYLKETAA AKFERQYLKETAAA KFERQYLKETAAAK FERQYLKETAAAKF ERQYLKETAAAKFE RQYLKETAAAKFER QYLKETAAAKFERQ YLKETAAAKFERQY L

Sequencing by MS/MS

For unambiguous sequencing all peptide bonds should be

broken

…-CHR – C(O) – NH – CHR’-…

Polypeptide backbone fragmentation

b

y

c

z

a

x

CollisionallyActivatedDissociation (CAD)

ElectronCaptureDissociation (ECD)

1960s, 1990s

1998ElectronDetachmentDissociation (EDD)

ElectronTransferDissociation (ETD)

2004

2004

InfraredMultiphotonDissociation (IRMPD)

1960s, 1995

157 nmUV Photodissociation

Metastable-atomInduced Dissociation(MAID)

2004

2005

ECD spectrum of 11+ ions from bovine ubiquitin

Problem of methods based on MS/MS identification

- Sensitivity lost –informative are only MS/MS spectra, whose intensity is at least ~10-fold lower than intensity of MS spectra

- There is no possibility to detect all peptides in one run

- Extra time for fragment spectra measurements causes longer chromatography time (application of UPLC is questionable for some types of MS instruments)

The other possibility in proteomics – usage of high mass measurement

accuracy mass spectrometry

(From Alan Marshall NHMFL)

Линейная ионная ловушка

FTMS Data

Магнит7 T

Электронная пушка

ИК-лазер

Linear ion trap

IR laser

Electron gun

Magnet

Ion cyclotron resonance mass spectrometer can measure masses with sub ppm accuracy

Other mass spectrometers with high accuracy of mass measurements are available nowOrbitrapsQ-TOFs…….

Mass accuracy 1-2 ppm (intern. calib.), 5 ppm (extern. calib.)Resolution 20 000-60 000 FWHM Rate of mass spectra measurements >20 Hz

BRUKER micrOTOF-QII

At accuracy level of 1 ppm elementary composition of peptide with mass up to 600 Da and amino acid composition of peptide with mass up to 500 Da could be determined almost unambiguously

It is not enough for peptide identification!

.

If we are using liquid chromatography (LC) or

Capillary electrophoreses (CE) we have another tag

- LC retention time or CE retention time

Accurate mass tag together with retention time Can identify peptide practically unambiguously!

Accurate mass tag retention timeDick Smith group (PNNL)

RT: 46.10 - 80.40

48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80

Tim e (m in)

0

20

40

60

80

100

120

0

20

40

60

80

100

120

0

20

40

60

80

100

120

Re

lativ

e A

bu

nd

an

ce

0

20

40

60

80

100

120

55.89

60.7364.54

65.9757.46 75.4258.0246.69 49.23 49.88 69.41 72.7954.17 68.8162.5378.2976.66

55.66

60.45

64.35

57.23 65.7857.9149.02 75.2446.48 49.68 55.05 68.6551.57 69.41 72.6166.7762.37

78.1476.47

55.65

60.64

64.33

65.9357.2557.9446.56 49.09 75.2669.2351.54 55.18 68.73 69.52 72.6162.38

78.1776.43

55.90

60.93

64.46

58.17 66.16 75.4246.66 49.97 60.13 68.81 72.7654.27 69.3551.5847.48 64.00 78.4474.20 76.56

NL: 1.07E6

Base Peak F: FTMS + p ESI Full m s [ 350.00-2000.00] MS urine_1-5_0-1ul_150m in

NL: 1.44E6

Base Peak F: FTMS + p ESI Full m s [ 350.00-2000.00] MS urine2nd

NL: 1.83E6

Base Peak F: FTMS + p ESI Full m s [ 350.00-2000.00] MS urine3thd

NL: 5.89E5

Base Peak MS urine4th

LC reproducibility-Agilent 1100LC reproducibility-Agilent 1100

Thus, there is a possibility in bottom-up approach to proteomics is to create using MS/MS a database for accurate mass tags and retention times as a reference base for fast quantitative measurements ofproteins and peptides concentration in a sample

VGLQR YVQLR SLR

450 500 550 600 650m/z

522.5 525.0m/z

LC- FTICR

Accurate measured mass: 1568.8768

Validated accurate mass tag (SLTLGIEPVSPTSLR)

...TGLYCESQTPRSLTLGIEPVSPTSLRVGLQRYVQLRSLR ...

…TGLYCESQTPR SLTLGIEPVSPTSLR

trypsinolyses

Fragment (463-477) from Vasorin

Putative mass tag from Homo Sapiens: SLTLGIEPVSPTSLRCalculated mass (1568.8773)And measured retention time

200 600 1,000 1,400 1,800m/z

y9

y8

b10

y7

b9b8

y6

y12

y10

y11b12

b6y5

b7

b11

y13b14b13

y4

LC-MS/MS (e.g. with ion trap)

identification

validation

Vasorin (Homo Sapiens protein)

FT ICR

I.Boldin, E.Nikolaev

ASMS May 2010Dynamicaly harmonized FT ICR cell

Pressure limited(practically unlimited mass resolution)

0 40 80 120 160 t (s)-1.5

-1.0

-0.5

0.0

0.5

x103

1.0

I(a.u.)

0 40 80 120 160 t (s)-1.5

-1.0

-0.5

0.0

0.5

x103

1.0

I(a.u.)

609.284060

609.2834 609.2838 609.2842 609.2846 m/z

R = 22,000,000

609.284060

609.2834 609.2838 609.2842 609.2846 m/z

609.284060

609.2834 609.2838 609.2842 609.2846 m/z

R = 22,000,000

Reserpine, Resolving Power 22,000,000 without apodization, 180 s transient

1072.50

1127.02

1166.53

1208.92

1231.27

1278.60

1303.64

1356.82

1414.48

1445.22

1510.87

1582.751621.38

1661.841704.40

1796.58

1100 1300 1500 1700 1900 m/z

1.0

2.0

x108

3.0

BSA, 0.3mg/ml, 100scans accumulated, accumulation time in collision cell 50ms (7 Tesla)

M Hn+

1384.246

1384.3701384.454

1384.495 1384.641

1384.767

1384.829

1384.9331384.975

1385.058

1385.142

1385.246

1385.371

1385.4961385.517

1385.6211385.704

1385.7871385.870

1385.9751386.0791386.162

1386.267 1386.4131386.433 1386.5791386.6411386.767

1386.8911386.9741387.1001387.225

1387.329

1387.350

1387.5171387.5791387.642

1387.726

1387.830 1387.9961388.079

1388.1631388.288

1388.372

1388.4761388.5591388.622

1388.7471388.851

1388.9541389.039 1389.2261389.3091389.3501389.475 1389.6431389.7671389.810 1389.9981390.081 1390.2661390.288 1390.415 1390.5791390.581 1390.809 1390.9981391.080 1392.2931392.309 1393.1061393.210 1413.847 1413.997 1414.1881414.2731414.3801414.486 1414.6141414.6991414.784 1414.996 1415.209

1384.6 1385.1 1385.6 1386.1 m/z

1.0

2.0

x108

22s

R = 1.3*106

BSA (65 kD) high resolution mode on 7 Tesla magnet

1208.9803

1208.72 1208.77 1208.82 1208.87 1208.92 m/z

0.5

1.0

1.5

2.0

x108

R = 0.9*106

Nb3SnCoils

NbTiCoils

21 Tesla FT-ICR Magnet

Field Center to Flange600 - 1100 mm

110 mm Bore

Current Leads, Cryocooler, and

Quench relief for Zero-Loss

2.2 °K Cryostat

D. Markiewicz, NHMFLT. Painter, NHMFLJ. Miller, NHMFLY. S. Choi, KBSI

Slide from Alan Marshall

FT MS

ESI Q-TOF

ESI TOF

Lab

Lab

Clinic

Accurate mass tag retention time approach

The most attractive is human plasma, which contains practically all proteins (around 20000 non modified forms)

Human Proteome Detection and Quantitation Project:hPDQ

N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson, Christoph H.Borchers, Amanda G. Paulovich, Scott D. Patterson, Michael Gillette, Ruedi Aebersold and Steven A. CarrMol Cell Proteomics Jan.2009

Proteins in bloodN. Leigh Anderson‡ and Norman G. Andersn

Protein concentrations are different by 11 orders of magnitude!!!There is no method to solve this analytical problem!

The main task is searching for proteinbiomarker of early stages of diseases

Alzheimer’s disease is a progressive brain disorder of elderly people that gradually destroys a person’s memory and ability to learn, reason, make judgments, communicate and carry out daily activities.

Alois Alzheimer (1864-1915)

1906 - 2006

Alzheimer disease

tangles

Plaques

Aβ – Amyloid A 1-42,

Beta-amyloid peptide

The main component of Alzheimer’s plaques (1984)

Sequenced in 1987

1DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42

Anomalous accumulation of Beta-amyloid in the form of polymeric aggregates (plaques, tangles) causes Alzheimer

• Why normal protein (monomeric Beta-amyloid) aggregates to form pathogenic plaques?

• What molecular event is triggering this process?

• Which part of the molecule is subjected to changing?

• How to detect these changes?

Questions to answer

Pro19

Substitution by prolineAbolishes fibril formation

Met35(O)Oxidation may be Important for toxicity and/or oligomerization

Asp7

Isomerized by 75%In plaques

Essential residues for self-association

Primary structure elements controlling Aβ oligomerization

The goal is to develop mass spectrometric methodology to distinguish peptides containing

different isomeric forms of individual amino acids and to apply this methodology to fragments

of Alzheimer disease Beta-amyloid

f

ECD of 1-16 Аβ

z10

z10

z9

z9

Z10 -57(Cα-Cβ bond destruction)

c9

c9

y9

C

Aβ 1-16(isoAsp7)

Aβ 1-16 (Asp7)

Distinguishing aspartate/iso-aspartate in Aβ – Amyloid by ECD

Y10

1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0102030405060708090

1000

102030405060708090

100

Rela

tive

Abun

danc

e

1198.45

1585.55

1349.451448.55

1722.73

1585.55

1349.451448.45

1220.271722.45

m/z

B6

700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 8600

102030405060708090

1000

102030405060708090

100

Rela

tive

Abun

danc

e

798.36 819.91

756.36

811.36 861.82

847.91

793.36

819.91

861.91847.91811.36

793.36756.27

Iso-aspartate

aspartate

aspartate

776.27

Y6 - H2O

Y6 - H2OY6

B11 B12 B13

B14

B10

DAEFRH DSGYEVHHQK b6

y10

CID

Iso-aspartate

B6+H2O

Distinguishing aspartate/iso-aspartate in Aβ – Amyloid by CID

Quantitative analyses

1020 1040 1060 1080 1100 1120 1140 1160 1180 1200 1220 1240

m/z

0

50

100

0

50

100

0

50

100

0

50

100

Re

lativ

e A

bu

nd

an

ce

0

50

100

0

50

100

0

50

1001237.53

1067.52

1182.551017.44

1198.561083.541047.49 1138.561030.45

1185.51

1234.251112.90

1059.40

1237.53

1074.461182.54

1017.44

1198.561083.531047.49 1138.551125.551020.67

1150.821100.60

1237.53

1067.521182.55

1017.44

1083.54 1198.561114.001047.49 1125.55

1061.17

1237.53

1074.47

1182.551017.45

1083.54 1125.551047.49 1198.571029.67 1213.811171.70

1237.53

1074.47

1182.551017.44

1125.551083.54 1198.571029.67 1163.481148.47 1210.531111.66

1237.53

1074.46

1182.541017.44

1125.551198.561083.531030.45 1047.49

1185.56

1112.59 1132.55

1059.67

1164.54

1237.53

1074.46

1067.52 1182.541017.44

1125.551083.54 1198.561030.45 1047.49 1104.34 1227.29

1063.07

NL: 1.04E3

Asp_ECD666.5_FT#1 RT: 25.09 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 8.13E2

10isoasp-90asp_ecd666.5_ft#1 RT: 53.31 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 7.30E2

15isoasp-85asp_ecd666.5_ft#1 RT: 16.50 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 3.87E2

30isoasp-70asp_ecd666.5_ft#1 RT: 29.90 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 7.22E2

asp-isoasp_1-1_ecd666.5_ft#1 RT: 131.70 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 1.54E3

70isoasp-30asp_ecd666.5_ft#1 RT: 43.08 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

NL: 1.01E3

isoasp_ecd666.5_ft#1 RT: 59.86 AV: 1 T: FTMS + p ESI Full ms2 666.50@0.00 mpd@4.00 [ mpd@180.00 -mpd@2000.00 ]

z10-57 z10 c10

z9 c9c8

Relative abundance of 1125.55(z10-57) peak(reference peak 1182.55(z10))

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 20 40 60 80 100

concentrationab

unda

nce

ratio

ECD mass spectra of six Аβ1-16(α-Asp) and Аβ1-16(β-Asp) peptide mixtures with different relative concentrations

Our recent results on detection of Aβ1-16, extracted from human blood

Prototype of molecular diagnostics method

• Extraction of Aβ fraction (~ 500 ng) from 5 ml of human plasma

• Preparation and extraction of Aβ1-16 probe from Aβ pool

• MS analysis of isoD7- to D7- Aβ1-16 ratio in the probe

The next goal is to see this peptide in urine!

42

Body liquids available noninvasively

Exhaled breath condensateUrineSalivaTearSweat

Noninvasive diagnostic of human breath system by mass spectrometry monitoring

of exhaled breath condensate

Breath condenser ECoScreen Jaeger from

VIASYSHealthcare (Germany)

KERATINS in individual EBC of young healthy nonsmoking donors

Other proteinsin individual EBC of young healthy nonsmoking donors

Proteins overexpressed in samples of patients with COPD (Chronic obstructive pulmonary disease) and pneumonia

COPD (n = 17) pneumonia (n = 13)1. Proteins of cytosceleton

Keratins 3, 4, 8 Keratins 4/13, 7/19, 8/18 and 15

Junction plakoglobin Hornerin Desmoplakin

2. Proteases inhibitors

Cystatin А Cystatin А Kininogen-1 Kininogen-1

Cystatins В, МAlpha-1-antitrypsin

3. Other Osteopontin Cytoplasmic actin

Monitoring of exhaled protein composition after human lung transplantation

Before surgery (artificial lung ventilation)

1st month after surgery 15 months after surgery

Pure protein spectrum because of disturbance of breathDermcidin, Keratin 9, Lysozyme, Ubiquitin

Allograft adoptation and medical treatment Annexin 1, Proteinases inhibitor,Bleomicine-hydrolase, keratin 8Damaged epithelium removal Desmosomal proteins (desmoglein, desmoplakin)Epithelium healing Hornerin, filaggrin

“Normal” proteinsDermcidin, “normal” keratins, Cystatin A, Ubiquitin

Analyses of urine proteom

SickHealthy

Urine is available in large quantities – ideal analyte for noninvasive diagnostic.

Possibility of biomarker discovery is attracting big attention.1500 proteins (from Mann’s group Adachi et al. Genome Biology 2006, V7, 9, R80) ; 2,362 proteins (Kentsis , A. et al. Proteomics Clinical Applications 2009, 3, (9), 1052-1061).

Three fractions of voided urine is under investigation

1.Proteome (masses of 3-60 kDa)

2.Peptidome (masses of 0.8-17 kDa)

3.Exosoms (50-90 nm vesicles secreted by a wide range of cell types)

Before use some proteins as biomarker we need to know its temporal variability and polymorphism(how different is its concentration in body liquids of different individuals)

To clarify this we need to investigate proteomes of hundreds of healthy individuals

Two kinds of sample donors

People “from street” (blood donation center)and

people in “special conditions”.

Decision to include a person to the study group

Current control for urogenital and other pathology including kidney

pathology, prostatitis, arterial hypertension, diabetes

Analysis of archival information from medical records

General blood analysis

Examination of internist

Blood pressure measurement

Control for treatment with diuretics and excessive consumption of fluids

For “people from street”

For “healthy people data base” subsetwe need urine samples from persons under well controlled diet and having healthy lifestyle?

In this case we can test urine temporal variability and polymorphism

Those are people participating In long term isolation experiments in the frame of space research programs. April- July 2009. March 2010 + 500 days. (The Institute for medical & biological problems RAS)

Ground based experimental facility

April- July 2009

Sample concentration Amicon Ultra Ultracel-15 3 k

Desalting and major protein removal

Urine collection

Centrifugation

LC MS analyses

Carboxymethylation and trypsinolyses

Database: IPI.Human v.3.52Parent Tolerance: ± 5.0 PPM (Monoisotopic)Fragment Tolerance: ± 0.50 Da (Monoisotopic)Fixed Modifications: Carbamidomethyl (C)Variable Modifications: Oxidation(M) Digestion Enzyme: TrypsinMax Missed Cleavages: 2Instrument type: Ion-trap

Search engine: Mascot

What is in the DB (Structured Query Language database)

• Run, in which this peptide was identified• Peptide sequence• What protein does this sequence belong to• Mascot score• Modifications• Measured mass• Theoretical mass• Measured charge• RT, when the peptide began to elute from the column• RT, when the peptide finished elution

Our statistics of the collected AMT tags in the long term isolation experiment

447 LC-MS (liquid chromatography coupled with mass spectrometry) runs totally:

among them

25 samples from each of 6 volunteers have been collected during105 days of isolation experiment.

The number of peptides in the database 3468The number of urine proteins in the database 1055

443 core proteins (all patients have them in their urine)

Smokers (41 sample) and non-smokers(46 samples)

Peptides Proteins

Total 2758 840

Current statistics of urinary proteome database for ordinary healthy people

Current statistics of urinary proteome database

233 LC-MS (liquid chromatography coupled with mass spectrometry) runs totally: 102 with samples from smokers, 131 with samples from non-smokers.

Using all peptides

Peptides Proteins

Non-smokers 2527 762

Smokers 1893 627

Total 2758 840

Influence of life stile on urine proteome

Smokers vs. non-smokers urine proteome

Using all peptidesPeptides Proteins

Non-smokers 2527 762Smokers 1893 627Total 2758 840

Peptides Proteins

785492132311662865

40% 35%

Using all peptidesPeptides Proteins

Odd 2232 445Even 2306 467Non-smokers 2535 506

Peptides Proteins

61406493032003229

20% 21%

Using all peptidesPeptides Proteins

Selection1 1723 365Selection2 1588 337Smokers 1894 400

Peptides Proteins

35302633061417171

25% 25%

!!

!!!

!

Differences in the numbers of observed proteins participating in particular biological process in urine of smokers and

nonsmokers

Transport, homophilic cell adhesion, lipid metabolic process, inflammatory response, innate immune response, epidermis development, defense response

!

This type of proteome analyses should be personalized !!

Quantitative analyses by 18O labeling

25 25

25

Ind

ivid

ual

no

n-l

abel

ed s

amp

les

Pool of labeled Pool of non- labeled

25 I

nd

ivid

ual

lab

eled

sam

ple

s

MS

C:\

RT: 69.40 - 87.80

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Time (min)

0

20

40

60

80

100

0

20

40

60

80

10076.66

80.0974.97

76.7880.21

76.5774.88 86.1181.4378.59

80.0071.17 78.53 86.9072.80 81.5778.7375.99 85.9972.69 84.6582.53 83.1370.85 73.47 74.80 78.44

76.74

80.0174.96

86.0476.8881.29

79.8971.40 78.5575.10 86.8079.3172.8371.46 76.07 80.1877.00 82.31 82.4373.9472.48 84.7971.26 80.4269.45 77.73 87.41

NL:

1.43E6

Base Peak MS

53_1-10_1ul

NL:

1.36E6

Base Peak MS

53_o18_1-

10_1ul

574 575 576 577 578 579 580 581 582

m/z

0

20

40

60

80

100

0

20

40

60

80

100

575.31

z=2

575.81

z=2

576.32

z=2

577.32

z=2

577.82

z=2

578.32

z=2

NL: 1.43E6

53_1-10_1ul#5161

RT: 76.66 AV: 1 T:

FTMS + p ESI Full

ms [

300.00-1600.00]

NL: 1.34E6

53_o18_1-

10_1ul#5122 RT:

76.74 AV: 1 T:

FTMS + p ESI Full

ms [

300.00-1600.00]

A List of Candidate Cancer Biomarkers for Targeted ProteomicsMalu Polanski and N. Leigh AndersonBiomark Insights. 2006; 1: 1–48. The Plasma Proteome Institute

list of 1261 proteins believed to be differentially expressed in human cancerAs an initial approach, we have selected a subset of the candidates based on a set of criteria including number of total citations, number of recent citations, proportion of recent citations, known plasma concentration (implying existence of an assay) and clinical use in any context. This subset of 260 candidates88 are detected in urine (Mann’s database) 75 (our database)

Our partners

Molecular & Cellular Proteomics 9:2424–2437, 2010.

Prof. Harald Mischak Mosaiques DiagnosticsGmbH, Mellendorfer Strasse 7–9, 30625 Hannover, Germany.

ROC curves for classificationof patient cohorts with “CKD pattern.”ROC analysis for CKD diagnosis of the training set and the test set after unblinding is shown. 85.5% sensitivity and 100% specificity

Peptide (800 to 17,000 Da) patterns distinguishingpatients with CKD from HC

230 patients 379 healthy

Samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases.

HPLC-MS run duration is about 1.5-2 hoursUPLC-MS duration is about 10-15 minutes

We need faster technology!!

• Efficient ion accumulation prior to IMS

• High mass accuracy, high dynamic range data acquisition system

• High IMS-TOF sensitivity due to ion trapping and multiplexing

ION MOBILITY SEPARATIONS IN HIGH THROUGHPUT ROTEOMICS: A NOVEL APROACH TO PROTEIN DETECTION AND IDENTIFICATION

PERIMENTAL PLATFORM

Mikhail BelovBiological Sciences DivisionPacific Northwest National Laboratory

TkdensityN

ZeK

bav _216

3

Mobility

K ELtdrift Drift time

Thermal diffusion-limited maximum resolution

Temporal spread

2ln16 Tk

LEZeR

bd

2

2

2sc

driftinit t

R

ttt

ION MOBILITY SPECTROMETRY (IMS)

ADDITIONAL ANALYTICAL PEAK CAPACITY DUE TO IMS

Only 3 features discerned without drift time dimension (*)

Conclusions

1.Accurate mass tag/retention time databases for human urine proteome and peptidome were created

2.Subset of the database contains data from healthy people leaving in long term isolation conditions under the same diet

3.The new approach is developed for rapid analyses of urine proteome using AMT/RT database

4.Significant difference in protein and peptide content of urine of people with kidney diseases has been found inside some groups of proteins and peptides responsible for particular pathways