Immunocapture Top-down Mass

Spectrometry Defines Components of HDL

for the Human Proteome Project

Julian Whitelegge Ph.D.

The Pasarow Mass Spectrometry Laboratory

NPI-Semel Institute for Neuroscience and Human

Behavior, David Geffen School of Medicine, UCLA

MBI, BRI, JCCC

11th November 2011, Agilent Technologies, La Jolla, California.

• Intact protein mass spectrometry

• Top-down high-resolution MS

• Immunocapture top-down MS

Zero-charge Molecular Mass Profile of bovine MIP

28,225.6

28303.8

28,343.3

28,112.2

28,042.2

Heterogeneity

Phosphorylation

Cysteinylation

C-terminal trimming

-L

-A

Avg. compound mass 28,225.20

Std. Deviation: 3.92

Mass calculated from bovine MIP

gene sequence = 28,223.1

D = 2.1 (0.0075 %)

Zero-charge Molecular Mass Profile of bovine MIP

28,225.6

28303.8

28,343.3

28,112.2

28,042.2

Heterogeneity

Phosphorylation

Cysteinylation

C-terminal trimming

-L

-A

Avg. compound mass 28,225.20

Std. Deviation: 3.92

Mass calculated from bovine MIP

gene sequence = 28,223.1

D = 2.1 (0.0075 %)

Unusual

Souda P, Ryan CM, Cramer WA, Whitelegge J., Methods. 2011, in press

as translated from ORF 15936

minus Met1 15805

plus N-acetyl 15847

1 disulfide/2 free thiol 15845

plus Cu/Zn 15172

dimer 30344

Human Cu-Zn Superoxide Dismutase (SOD1)

More usual

Joan Selverstone Valentine

as translated 15936

minus Met1 15805

plus N-acetyl 15847

1 disulfide/2 free thiol 15845

plus Cu/Zn 15172

dimer 30344

Human Cu-Zn Superoxide Dismutase (SOD1)

Prudencio M, Durazo A, Whitelegge JP, Borchelt DR. Hum Mol Genet. 2010 Dec 15;19(24):4774-89.

‘An intact protein mass spectrum defines the native covalent

state of a gene product and its heterogeneity’

(a molecular profile)

‘An intact protein mass spectrum defines the native covalent

state of a gene product and its heterogeneity’

(a molecular profile)

One gene product shaped by several other gene products

and its environment.

The intact mass profile reflects one gene product in the

context of a complex system.

Intact mass profiles are dynamic.

PsbH - Relative Expression Data

Post-translational modifications

PsbH - Relative Expression Data

Post-translational modifications - phosphorylation

PsbH - Relative Expression Data

Light-induced changes in PsbH

expression are monitored by LC-MS

- a second phosphorylation site

- appearance of +32 Da adducts

+16, oxidation independent of phosphorylation

+16 Da

PsbH - Relative Expression Data

Light-induced changes in PsbH

expression are monitored by LC-MS

- a second phosphorylation site

- appearance of +32 Da adducts

Linkage of oxidative modification with phosphorylation ??

+32 Da

Gómez SM, Nishio JN, Faull KF, Whitelegge JP. (2002) Molecular and Cellular Proteomics 1, 45-59.

Simple logic: on/off toggle

Advanced logic: if x on, and y on then ….

Histone code, David Allis

Technical hurdles:

Membrane proteins

Sample requirements

Technical hurdles:

Membrane proteins

Large polytopic integral membrane proteins

Sample requirements

Technical hurdles:

Membrane proteins

Large polytopic integral membrane proteins

Sample requirements

No PCR for proteins

Intact mass proteomics with

integral membrane proteins

Integral membrane proteins (30 % of the proteome)

are insoluble in most of the solvent systems

traditionally used for mass spectrometry.

Develop new chromatography systems involving volatile

aqueous and organic solvent systems that are both

compatible with ESI-MS and solubility of membrane

proteins.

PROBLEM

SOLUTION

CHROMATOGRAPHY OF MEMBRANE PROTEINS

IN

AQUEOUS/ORGANIC SOLVENT MIXTURES

Reverse-phase chromatography (Whitelegge et al, 1992; 1998)

Stationary phase: PLRP/S, 40 ˚C

Mobile phase 1:

A. 60 % formic acid, B. isopropanol

Mobile phase 2:

A. 0.1 % TFA in water, B. 0.05 % TFA, 50 % acetonitrile, 50 % isopropanol

(Tarr and Crabbe, 1984; Whitelegge et al, 2002)

Size-exclusion chromatography (Whitelegge et al, 1992; 1999)

Stationary phase: Tosoh G2000SW

Mobile phase:

chloroform/methanol/1 % formic acid (4/4/1, v/v)

(Fearnley and Walker, 1996)

CHROMATOGRAPHY OF MEMBRANE PROTEINS

IN

AQUEOUS/ORGANIC SOLVENT MIXTURES

Reverse-phase chromatography (Whitelegge et al, 1992; 1998)

Stationary phase: Agilent PLRP/S, 40 ˚C

Mobile phase 1:

A. 60 % formic acid, B. isopropanol

Mobile phase 2:

A. 0.1 % TFA in water, B. 0.05 % TFA, 50 % acetonitrile, 50 % isopropanol

(Tarr and Crabbe, 1984; Whitelegge et al, 2002)

Size-exclusion chromatography (Whitelegge et al, 1992; 1999)

Stationary phase: Tosohaas G2000SW

Mobile phase:

chloroform/methanol/1 % formic acid (4/4/1, v/v)

(Fearnley and Walker, 1996)

ESI-MS of intact polytopic integral

membrane proteins

LC-MS+

HPLC Reverse-phase or

Size exclusion

Electrospray-ionization mass

spectrometry (ESI-MS)

- MALDI-TOF for heterogenous proteins.

- Mass & Sequence tags for identification

- Post-translational modifications by MS-MS.

- Westerns

- Top-down/middle-down MS

Mass spectrum of intact protein

- monitor phosphorylation &

other covalent modifications

Protein sub-fraction

Fraction collector

Splitter

Sample requirements issue:

Miniaturization of chromatography

Micro-fluidics project

• Couple microfluidics to miniturized LC-MS

• Screen 1000 small molecules

• Stabilization of integral membrane proteins

via H-D exchange assay

• Less than 1 ug/reaction

• High-throughput (1000/wk)

• Clifton Shen, Libo Zhao, CNSI

Intact mass screening for substrates of enzymes that

perform post-translational modifications

• Intact protein mass spectrometry

• Top-down high-resolution MS

• Immunocapture top-down MS

Top-down high-resolution mass spectrometry

Traditionally Fourier-transform ion-cyclotron resonance MS

Kelleher et al introduce top-down protein mass spectrometry

Kelleher NL, Lin HY, Valaskovic GA, Aaserud DJ, Fridriksson EK, McLafferty

FW. Top-down versus Bottom-up Protein Characterization by Tandem High-

resolution Mass Spectrometry. J Am Chem Soc. 1999;121:806–812.

Zubarev, Kelleher and McLafferty introduce ECD

Zubarev RA, Kelleher NL, McLafferty FW. Electron Capture Dissociation of

Multiply Charged Protein Cations. A Nonergodic Process. J Am Chem Soc.

1998;120:3265–3266.

Top-down MSMS of small intact integral membrane proteins

QTOF – unit resolution

M. laminosus PetG - triply charged parent, 1352.5

Subunit Sequence MW

S.oleracea (calc.) (meas.)

PetG formylMIEVFLFGIVLGLIPITLAGLFVTAYLQYRRGDQLDL 4198.03 4196.32

NP054954 MIEVFLFGIVLGLIPITLAGLFVTAYLQYRRGDQLDL

PetM

A.thaliana

NAVGEIFKIAAIMNALTLVGVAVGFVLLRIEATVEEAE

NAVGEIFKIAAIMNALTLVGVAVGFVLLRIETSVEEAEAE

3972.70 3971.18

PetL formylMFTLTSYFGFLLAALTITSALFIGLNKIRLI 3478.24 3476.97

NP054953 MSTLTSYFGFLLAALTITSALFIGLNKIRLI

PetN formylMDIVSLAWAALMVVFTFSLSLVVWGRSGL 3197.84 3196.67

NP054925 MDIVSLAWAALMVVFTFSLSLVVWGRSGL

M.laminosus

PetG formylMVEPLLDGLV LGLVFATLGG LFYAAYQQY… NA 4057.4

PetM MTEEMLYAAL LSFGLIFVGW GLGVLLLKIQ GAEKE 3841.7 3841.0

PetL formylMILGAVFYIVFIALFFGIAVGIIFAIKSIKLI 3530.5 3530.4

PetN formylMEIDVLGWVALLVVFTWSIAMVVWGRNGL 3304.0 3303.6

1. Spinach chloroplast sequences from Schmitz-Linneweber,C., Maier,R.M.,

Alcaraz,J.P., Cottet,A., Herrmann,R.G. and Mache,R.The plastid

chromosome of spinach (Spinacia oleracea): complete nucleotide

sequence and gene organization. Plant Mol. Biol. 45 (3), 307-315 (2001)

Whitelegge et al, Mol. Cell. Proteomics 1, 2002.

A previously undiscovered RNA editing event ?

TOP-DOWN SEQUENCING OF INTACT SMALL SUBUNITS

BY TANDEM MASS SPECTROMETRY (MSMS)

c-subunit

c-subunit can be extracted into chloroform (proteolipid)

and purified by SEC with on or offline MS

First analyzed by top-down on QTOF

Whitelegge - TrAC Trends in Analytical Chemistry, 2005.

FT-ICR on M+5H+

Activated ion electron capture dissociation

(aiECD) provides expanded coverage in the

transmembrane domain compared with collision

activated dissociation (CAD)

Zabrouskov V, Whitelegge JP. (2007) J. Proteome Res.

2.5 ppm RMS

4.7 ppm RMS

Murine voltage-dependent anion channel (VDAC)

A role in the mitochondrial permeability transition (MPT)

Inhibition might be cardioprotective

Ujwal R. et.al. PNAS 2008;105:17742-17747

Beta-barrel transmembrane porin

An inhibitor of MPT ?

Intact VDAC protein profiles

allow rapid determination of

conditions for specific modification

High efficiency single modification

of VDAC with Ro 68-3400

Intact VDAC protein profiles

allow rapid determination of

conditions for specific modification

High efficiency single modification

of VDAC with Ro 68-3400

Specific modification ?

2nd Cys modified

Modified y-ions support C244mod

unmodified modified

Ryan CM, et al, Mol Cell Proteomics. 2010 9(5):791-803.

cr061610_Maduke_pH45_ppt_250um_MS_120sc #1 RT: 233.22 AV: 1 NL: 2.80E4

T: FTMS + p ESI w SIM ms [1440.00-1640.00]

1440 1460 1480 1500 1520 1540 1560 1580 1600 1620 1640

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

ela

tive

Ab

un

da

nce

1570.95

1621.53

1523.381478.63

1601.55

1551.481590.46

1542.29

1504.58

1496.84

1460.30

1609.94

1529.411454.16 1628.04

1577.26

1596.681561.061484.42

1466.12 1510.521442.07

1515.50

1471.971615.821580.12

1537.13

1566.99

1445.67 1491.51

1639.21

1632.60

Figure 1

Mass spectrum in range 1440-1640 m/zMultiply charged ions

Needs transformation to m

50 kD chloride channels, CLCec1

Merritt Maduke

50239 Da

cr061610_Maduke_pH45_ppt_250um _ii1571_10sc #1 RT: 235.53 AV: 1 NL: 8.15E4

T: FTMS + p ESI Full ms2 1571.00@cid0.00 [600.00-2000.00]

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

und

an

ce

1571.02

836.14 1466.49

1705.901593.28 1884.241800.17733.30 1489.961413.801049.96

1540.72

1306.61 1986.56659.62 1163.68 1238.71889.76774.72 946.24

cr0 61 61 0_Mad uke_ pH 45_pp t_ 250 um _i i15 71 _1 0sc #1 RT: 235 .53 AV: 1 N L: 8.1 5E4

T: FTMS + p ESI Ful l ms 2 1 57 1.00@cid0 .00 [600.00-2 00 0.0 0]

1570.0 1 570.2 1570 .4 1 57 0.6 1 570 .8 15 71.0 1 571 .2 15 71 .4 15 71.6 1 57 1.8 15 72.0 1 57 2.2 1 572 .4 1 57 2.6 1 572 .8 15 73 .0

m/z

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

10 0

Re

lative

Ab

un

da

nc

e

1 57 1.02157 0.89

15 71.05

15 71.11

1 570 .8 3

157 1.1715 70 .80

15 71 .2415 70 .7 7

15 71.27

15 70.711571.33

157 1.77

15 70 .67 157 1.461 571 .8 9

15 71.67

15 72.02157 0.63

15 72.0815 70 .24 1570 .4 6

1 572.36 15 72.481 56 9.93 1 572 .6 8

1 57 0.12

1 572 .9 5157 3.07

Figure 3

Ion isolation

Singly modified protein

1571

Unit resolution at 50,000 Da

Modification localized

• Intact protein mass spectrometry

• Top-down high-resolution MS

• The human proteome project and

immunocapture top-down MS

Human Salivary Proteome Project

(NIDCR)

• Define the secreted human salivary

proteome

• Salivary diagnostics

• Top-down identification of human Cystatins

The Cystatin family

Cystatin

Swiss-Prot

# amino

acids

Modification

Measured average

mass (Da)

Calculated monoisotopic

mass after

modification (Da)

Experimental monoisotopic

mass (FTMS)

(Da)

Delta (Da)

Delta (ppm)

RMSa

(ppm)

S

P01036

121

1-20 removed,

2 disulfide bonds

14186

14175.8005

14175.8569

0.0564

3.98

3.69

S1

P01036

121

1-20 removed, 2 disulfide bonds, phosphorylation

14266

14255.7668

14255.8567

0.0899

6.30

4.26

S2

P01036

121

1-20 removed,

2 disulfide bonds, 2 phosphorylation

14346

14335.7335

14335.8110

0.0775

5.40

4.75

SA

P09228

121

1-20 removed,

2 disulfide bonds

14347

14337.0014

14336.9856

0.0158

1.10

6.55

SN

P01037

121

1-20 removed,

2 disulfide bonds

14313

14303.2228

14303.1553

0.0675

4.72

5.00

SN (SNP)

P01037

121

1-20 removed, 2 disulfide bonds,

rs2070856 (P31L)

14328

14319.1187 14319.171b

0.0523

3.65 b

7.07 b

C

P01034

120

1-26 removed,

2 disulfide bonds

13345

13334.5969

13334.5829

0.0140

1.05

3.90

D (SNP)

P28325

114

1-28 removed,

2 disulfide bonds, rs1799841 (C46R)

13165

13154.4776

13154.4675

0.0101

0.77

2.47

D (SNP)

P28325

118

1-24 removed, 2 disulfide bonds, rs1799841 (C46R)

NDc

13596.7064

13596.7015

0.0049

0.36

0.35

Cystatin

Swiss-Prot

# amino

acids

Modification

Measured average

mass (Da)

Calculated monoisotopic

mass after

modification (Da)

Experimental monoisotopic

mass (FTMS)

(Da)

Delta (Da)

Delta (ppm)

RMSa

(ppm)

S

P01036

121

1-20 removed,

2 disulfide bonds

14186

14175.8005

14175.8569

0.0564

3.98

3.69

S1

P01036

121

1-20 removed, 2 disulfide bonds, phosphorylation

14266

14255.7668

14255.8567

0.0899

6.30

4.26

S2

P01036

121

1-20 removed,

2 disulfide bonds, 2 phosphorylation

14346

14335.7335

14335.8110

0.0775

5.40

4.75

SA

P09228

121

1-20 removed,

2 disulfide bonds

14347

14337.0014

14336.9856

0.0158

1.10

6.55

SN

P01037

121

1-20 removed,

2 disulfide bonds

14313

14303.2228

14303.1553

0.0675

4.72

5.00

SN (SNP)

P01037

121

1-20 removed, 2 disulfide bonds,

rs2070856 (P31L)

14328

14319.1187 14319.171b

0.0523

3.65 b

7.07 b

C

P01034

120

1-26 removed,

2 disulfide bonds

13345

13334.5969

13334.5829

0.0140

1.05

3.90

D (SNP)

P28325

114

1-28 removed,

2 disulfide bonds, rs1799841 (C46R)

13165

13154.4776

13154.4675

0.0101

0.77

2.47

D (SNP)

P28325

118

1-24 removed, 2 disulfide bonds, rs1799841 (C46R)

NDc

13596.7064

13596.7015

0.0049

0.36

0.35

Uniprot/Swiss-Prot is the annotated database

Uniprot/Swiss-Prot is the annotated database

Intact mass measurements define the human proteome

HUPO

• Human Proteome Project (HPP), announced 2010

• Mission Statement of HPP

The mission of the Human Proteome Project (HPP): “The

Human Proteome Project, by characterizing all 21 000 genes of

the known genome, will generate the map of the protein based

molecular architecture of the human body and become a

resource to help elucidate biological and molecular function and

advance diagnosis and treatment of diseases ”

• Top-down initiative on its way

Top-down community is gearing up to address the

challenge of the human proteome project (HPP)

How will we find lower abundance components in

complex mixtures ?

How will we find lower abundance components in

complex mixtures ?

Immunocapture

HUPO • Human Antibody Initiative

• The mission of the Human Antibody Initiative (HAI) aims to promote

and facilitate the use of antibodies for proteomics research. The

initiative consists of two separate activities; (1) the generation of a

catalogue of validated antibodies from many different sources and

(2) a protein atlas for the expression and localization of human

proteins in normal and disease tissue. The two separate activities

have as their primary deliverables to generate databases with free

public accessibility. The Antibody Resource database

(www.antibodypedia.org) is aimed to produce a comprehensive

catalogue of validated antibodies towards human proteins. This

initiative depends on input from a large number of academic groups

and commercial companies. The Protein Atlas initiative

(www.proteinatlas.org) is aimed to provide comprehensive and

annotated database of high-resolution images showing tissue profiles

in normal and cancer tissues. Both databases will be open to the

public without restriction (no passwords).

MARS column • Introduced for depletion of plasma for biomarker studies on flow-through

• Retained material could provide a wealth of information

• Single antibody column could be used for immunocapture of targeted protein

and its binding partners

• Proved the concept with anti-Apo A-I column in collaboration with Chris

Vanselow Ph.D. at Agilent Technologies

• First presented at 7th USHUPO, March 20 - 23, 2011 Raleigh Convention

Center, North Carolina. Immunocapture top-down mass spectrometry defines

components of HDL for the human proteome project. Julian Whitelegge,

Christopher M. Ryan, Li Jing, Puneet Souda, Kym Faull, Chris Vanselow

Other proteins in human serum Apolipoprotein A-I and partners

Enrichment of Apolipoproteins from human serum

by using antibody-based immunoaffinity

chromatography

Chris Vanselow (Agilent) Li Jing (UCLA)

Reverse phase LC-MS

61

Ion chromatogram

Contour

plot

Apo CIII

Apo AI

Apo AII

Apo AI+ acyl

Apo CI

Fraction 42, ion isolation of

peak1326 z=5 [M]=6626.5017

Apolipoprotein C-I

Human serum_111110LCMS_fraction42 ii1326_z5 #1 RT: 2.77 AV: 1 NL: 8.81E4T: FTMS + p NSI Full ms2 1327.50@cid0.00 [365.00-2000.00]

1323 1324 1325 1326 1327 1328 1329 1330 1331 1332

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

1327.1084

1327.3085

1326.7084

1327.5088

1327.7091

1327.9097

1326.3076

1328.1100

1328.31081325.68421323.9069 1324.88651322.8586 1329.6046 1330.3634 1332.5647

High-resolution top-down MS

Fraction 45, ion isolation of

peak1214 z=8

[M]=9706.5219

Apolipoprotein C-III-2

Human serum_111110LCMS_fraction45 ii1214_z8 #1 RT: 5.00 AV: 1 NL: 5.58E4T: FTMS + p NSI Full ms2 1215.50@cid0.00 [330.00-2000.00]

1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

1215.0746

1214.6993

1215.3248

1214.4485

1213.0747

1215.5747

1213.8159

1211.64341210.6420 1216.0498 1217.0695 1218.8285

Fraction 64, ion isolation

of peak969 z=29

[M]=28061.3983

Apolipoprotein A-I

Human serum_111110LCMS_fraction64 ii969 #1 RT: 8.90 AV: 1 NL: 9.97E4T: FTMS + p NSI Full ms2 969.80@cid0.00 [265.00-2000.00]

965 966 967 968 969 970 971 972 973 974 975

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

969.1605969.3316

968.9896

969.5038

968.6081

969.7799968.1562

967.1603970.0211

966.5729

965.8456970.6235 973.2462 975.1116972.4232971.6633 974.0004

Human serum_111110LCMS_fraction64 ii969 #1 RT: 8.90 AV: 1 NL: 9.97E4T: FTMS + p NSI Full ms2 969.80@cid0.00 [265.00-2000.00]

968.9 969.0 969.1 969.2 969.3 969.4 969.5 969.6

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

969.1605969.2292

969.2630

969.1267

969.0923 969.2970

969.3316

969.0581

969.3652

969.0236

969.4001

968.9896 969.4344

969.4689

969.5038968.9542969.5722

968.8866

HDL preparations from many different mammals have been

analyzed, in collaboration with Don Puppione Della Donna L, Bassilian S, Souda P, Nebbia C, Whitelegge JP, Puppione DL. Mass spectrometric

measurements of the apolipoproteins of bovine (Bos taurus) HDL. (2011) Comp Biochem Physiol

Part D Genomics Proteomics. Oct 12. PMID: 22056023

Puppione DL, Ryan CM, Bassilian S, Souda P, Xiao X, Ryder OA, Whitelegge JP. (2010) Detection

of two distinct forms of apoC-I in great apes. Comp Biochem Physiol Part D Genomics Proteomics.

5(1):73-9. PMID: 20209111

Puppione DL, Della Donna L, Laganowsky AD, Bassilian S, Souda P, Ryder OA, Whitelegge JP.

(2009) Mass spectral analyses of the two major apolipoproteins of great ape high density

lipoproteins.Comp Biochem Physiol Part D Genomics Proteomics 4(4):305-309. PMID: 20161347

Puppione DL, Donna LD, Laganowsky AD, Bassilian S, Souda P, Ryder OA, Whitelegge JP. (2009)

Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins.

Comp Biochem Physiol Part D Genomics Proteomics 4(4):305-9. PMID: 21298813

Puppione DL, Bassilian S, Souda P, MacDonald MH, Halgand F, Whitelegge JP. (2008) Mass

spectral analysis of the apolipoproteins on dog (Canis lupus familiaris) high density lipoproteins.

Detection of apolipoprotein A-II. Comp Biochem Physiol Part D Genomics Proteomics 3(4):290-6.

PMID: 20483223

Puppione DL, Yam LM, Bassilian S, Souda P, Castellani LW, Schumaker VN, Whitelegge JP.

(2006) Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection

of post-translational modifications. Biochim Biophys Acta. 1764(8):1363-71. PMID: 16876491

Puppione DL, Whitelegge JP, Yam LM, Bassilian S, Schumaker VN, MacDonald MH. (2005) Mass

spectral analysis of domestic and wild equine apoA-I and A-II: detection of unique dimeric forms of

apoA-II. Comp Biochem Physiol B Biochem Mol Biol. 142(4):369-73. PMID: 16230041

Puppione DL, Whitelegge JP, Yam LM, Schumaker VN. (2005) Mass spectral analysis of pig (Sus

scrofa) apo HDL: Identification of pig apoA-II, a dimeric apolipoprotein. Comp Biochem Physiol B

Biochem Mol Biol. 141(1):89-94. PMID: 15820138

Puppione DL, Fischer WH, Park M, Whitelegge JP, Schumaker VN, Golfeiz S, MacDonald MH.

(2004) Sequence of horse (Equus caballus) apoA-II. Another example of a dimer forming

apolipoprotein. Comp Biochem Physiol B Biochem Mol Biol. 138(3):213-20. PMID: 15253869

Reverse-phase LC-MS+

Contour plot

+266 +532 A-I

A-II

The delta 266 Da (264-266) is more highly retained in

reverse-phase chromatography. Such a change in retention

is generally due to increased hydrophobicity. This is

consistent with fatty-acylation.

Hypothesis:

delta 266 Da is due to modification with fatty acids such as

stearate and oleate (264).

Quantitative separation of fatty-acylated human apo A-I

Modified

Top-down identification of modification site(s) - modified

Insufficient sequence coverage, 266 Da modification localized

Middledown approach - Frederic Halgand

Use CNBr to generate a set of smaller pieces to be

resolved on the mass spectrometer without further

sample handling.

Plate-forme Protéomique Biogenouest

Campus de Beaulieu - Bâtiment. 24 - CS2407

263 Avenue du Général Leclerc

35042 Rennes Cedex, France

CNBr has yielded a modified peptide.

Due to neutral loss, only fragments that carry the

modification can be used to localize it.

S210 modification is supported by smallest ions that carry the

modification, and that bracket the site.

PTM mass 266.26095

ECD on two different parents support stearoylation of Ser210

CONCLUSIONS

1. Intact protein mass profiles provide a unique

perspective on the human proteome

2. High-resolution top-down mass spectrometry is the

technology of choice

3. Immunocapture, a vital technology

4. Middle-down approaches are often required for precise

localization of modification sites – very accessible to

modern QTOF technology

5. Full understanding of PTM dynamics will require

integrated multi-faceted approaches

The Future

• All human proteins defined by primary

structure, intact mass measurements

• Immunocapture

• Variation across mitochondria, other

organelles, cells, tissues documented

• Dynamics measured and modeled in human

systems

The Pasarow Mass Spectrometry Laboratory

• Kym Faull Ph.D.

• Puneet Souda MBA.

• Chris Ryan Ph.D.

• Upendra Kar Ph.D.

• Li Jing Ph.D.

• Sara Bassilian (VA)

Funding

• Ping (PPG, NHLBI Center)

• Valentine PPG

• McBride U01

• Cramer P50

• Cohen, Reeve R01s

• Wong U01, T32

• Whitelegge, Maidment, Krogstad R21s

• Loo (HEI)