Tài liệu Báo cáo khoa học: A functional polymorphism of apolipoprotein C1 detected by mass spectrometry docx

This study reports the first case of a structural variant of ApoC1 as well as some protein properties that suggest the functional significance of this residue change.. Briefly, peak identifi

by mass spectrometry

Matthew S Wroblewski1, Joshua T Wilson-Grady1, Michael B Martinez1, Raj S Kasthuri2,

Kenneth R McMillan3, Cristina Flood-Urdangarin4and Gary L Nelsestuen1

1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

2 Department of Medicine, University of Minnesota, Minneapolis, MN, USA

3 American Indian Community Development Corporation, Minneapolis, MN, USA

4 St Mary’s Health Clinics, St Paul, MN, USA

Apolipoprotein C1 (ApoC1) is a component of

very-low-density lipoproteins (VLDLs), intermediate classes,

and high-density lipoproteins (HDLs) It has several

potential functions It helps to maintain HDL structure

and activates plasma lysolecithin acyltransferase It is

also able to modulate the interaction of apolipoprotein

E with b-migrating VLDLs and inhibit binding of

b-VLDL to low-density lipoprotein receptor-related

protein [1,2] It is implicated in regulation of several

lipase enzymes [3–5] An N-terminal 38-residue form of

ApoC1 is able to inhibit cholesterol ester transferase

[6] ApoC1 accounts for inhibition of cholesterol ester

transferase by HDL [7] Thus, ApoC1 has a number of

potential functions that may be important in vivo

Known variants of the ApoC1 gene are limited to

un-translated regions of the gene, synonymous mutations

of the coding sequence and a number of variants of the intron regions of the gene (NCBI database for ApoC1)

An important functional variant is found in the promo-ter region where complex factors [8,9] may link ApoC1 expression levels to familial dysbetalipoprotemia, car-diovascular disease, and Alzheimer’s disease [10–12] Overexpression of human ApoC1 in the mouse produ-ces a hyperlipidemic condition [4,13] with possible beneficial effects for diabetes [14,15] Hyperlipidemia may result from increased inhibition of b-VLDL bind-ing to the receptor and reduced clearance of VLDLs from the circulation Variants of ApoC2 and ApoC3 have been linked to metabolic disease [16–18] This study reports the first case of a structural variant of ApoC1 as well as some protein properties that suggest the functional significance of this residue change They

Keywords

apolipoprotein C1; mass spectrometry;

polymorphism; protein–lipid contact surface

Correspondence

G L Nelsestuen, 6–155 Jackson Hall,

321 Church St SE, Minneapolis, MN 55455,

USA

Fax: +612 625 2163

Tel: +612 624 3622

E-mail: nelse002@umn.edu

(Received 7 July 2006, revised 16 August

2006, accepted 18 August 2006)

doi:10.1111/j.1742-4658.2006.05473.x

A survey of plasma proteins in approximately 1300 individuals by MALDI-TOF MS resulted in identification of a structural polymorphism

of apolipoprotein C1 (ApoC1) that was found only in persons of American Indian or Mexican ancestry MS⁄ MS analysis revealed that the alteration consisted of a T45S variation The methyl group of T45 forms part of the lipid-interacting surface of ApoC1 In agreement with an impact on lipid contact, the S45 variant was more susceptible to N-terminal truncation by dipeptidylpeptidase IV in vitro than was the T45 variant The S45 protein also displayed greater N-terminal truncation (loss of Thr-Pro) in vivo than the T45 variant The S45 variant also showed preferential distribution to the very-low-density lipoprotein fraction than the T45 protein These prop-erties indicate a functional effect of the S45 variant and support a role for residue 45 in lipid contact and lipid specificity Further studies are needed

to determine the effects of the variant and its altered N-terminal truncation

on the metabolic functions of ApoC1

Abbreviations

ApoC1, apolipoprotein C1; ApoC2, apolipoprotein C2; ApoC3-0, ApoC3 that does not contain a carbohydrate chain; ApoC3-1, ApoC3 with a GalNAc-Gal-sialic acid carbohydrate chain; ApoC3-2, ApoC3 containing the carbohydrate of ApoC3-1 plus an additional sialic acid residue; DPPase, dipeptidylpeptidase IV; HDL, high-density lipoprotein; TTr, transthyretin; VLDL, very-low-density lipoprotein.

also suggest approaches that might be used to

deter-mine the role of N-terminal truncation of ApoC1

Results

Profile analysis

The MALDI-TOF mass spectrometer detects m⁄ z

val-ues that generally equate to protonated molecules

Figure 1A shows an overview of the plasma protein

profile Briefly, peak identification was accomplished

by comparison of m⁄ z values with those of known

plasma proteins, Edman degradation of the entire

sam-ple with observed removal of mass appropriate for the

expected N-terminal residue of each component, and⁄ or C-terminal degradation by carboxypeptidase with removal of mass appropriate for the expected res-idues of each protein An additional approach used protein reactivity with disulfide reagents such as dithio-threitol and iodoacetamide, with quantification of Cys

by detection of mass change of a peptide after reduc-tion and alkylareduc-tion These and other approaches have been described previously [19], and components that are important to this study are summarized in the legend to Fig 1 Figure 1B shows an expanded view of the ApoC1 proteins from this individual who showed

a double peak for each of the two forms of ApoC1 This double peak pattern was highly unusual and was not observed in over 1000 individuals with ancestry of Europe, Africa and Asia Forty-four instances of this pattern were found among 314 people who identified themselves as having American Indian and⁄ or Mexican ancestry Fig 1C shows an expanded view of the transthyretin (TTr) components from the profile of a different individual who displayed a double peak pat-tern that suggested a common polymorphism of TTr Many plasma proteins are present in multiple forms For example, ApoC1 is present as the full-length pro-tein (m⁄ z ¼ 6632) and as a truncated form lacking N-terminal Thr-Pro [19,20] (m⁄ z ¼ 6434, Fig 1) TTr exists as an unmodified protein (m⁄ z ¼ 13762) and as

a form that is disulfide-linked to cysteine (m⁄ z ¼

13881, Fig 1) Polymorphisms appear as a double peak for each form of a given protein The peaks differ

by the mass change produced by the amino-acid sub-stitution Figure 1C shows the example of a commonly observed double peak for TTr with a second compo-nent that is 30 atomic mass units (amu) higher than the common form This double peak for TTr was observed in  13% of samples and may represent a common G6S variant [21]

Of greater interest were the unusual components occurring at 14 amu below full-length and truncated ApoC1 All examples of the pattern in Fig 1 showed the same general characteristics That is, the peak occurring 14 amu below full-length ApoC1 was much less intense than the peak for full-length ApoC1; the peak at 14 amu below truncated ApoC1 was equally

as intense as or slightly more intense than the peak from truncated ApoC1 It is possible that all instances

of this novel profile feature pattern arose from the same modification and were genetically determined

Structural change ApoC1 from a person with the double peak profile in Fig 1B was isolated as described in Experimental

13762

m/z

6632

6618 6420

6434

6632

13881

13762 13792

13881 13911

TTr

A

C

9422

9713 8915

Fig 1 MALDI-TOF profile of plasma from an individual containing

the unusual profile (A) The profile from m ⁄ z ¼ 6000–15 000

Sev-eral peaks are labeled with their m ⁄ z values (B) Expanded view of

the ApoC1 portion of the profile in (A) Important components of

the profile include: ApoC1 (m ⁄ z ¼ 6632) and its truncated form

(m ⁄ z ¼ 6434), an ApoC1 variant (m ⁄ z ¼ 6618) and its truncated

form (m ⁄ z ¼ 6420) (C) Expanded view of the TTr portion of the

profile from a person who displayed a commonly observed double

peak for TTr The m ⁄ z values and suggested protein identities are:

13762, the common form of TTr; 13881, the common form of TTr

disulfide-linked to cysteine; 13792, a variant form of TTr that may

consist of G6S change; 13911, the variant protein that is

disulfide-linked to cysteine.

procedures, digested with Glu-C protease, and the

pep-tides subjected to MS The peptide mass fingerprint

from MALDI-TOF MS showed m⁄ z values

corres-ponding to all eight theoretical peptides plus the

peptide of the truncated protein (residues 1–13,

TPDVSSALDKLKE, theoretical mass¼ 1402.7 amu;

residues 3–13 (the truncated protein), 1204.7 amu;

resi-dues 14–19, FGNTLE, 680.3 amu; resiresi-dues 20–24,

DKARE, 618.3 amu; residues 25–33, LISRIKQSE,

1073.6 amu; residues 34–40, LSAKMRE, 834.4 amu;

residues 41–44, WSFE, 568.2 amu; residues 45–51,

TFQKVKE, 879.5 amu; residues 52–57, KLKIDS,

703.4 amu) Only peptide 45–51 showed a second peak

that was 14 ± 0.1 amu lower (Table 1)

The parent peptide (m⁄ z ¼ 879.467, residues 45–51

of ApoC1) provided four potential mutations that

would result in loss of 14 amu (T45S, Q47N, K48N,

K50N) Cleavage by Glu-C protease established that

the C-terminal Glu was unaltered The observed mass

difference (14.003 amu, Table 1) represented a

60 p.p.m error for peptides of m⁄ z ¼ 879 and 865 that

differ by a K⁄ N mutation (theoretical difference ¼

14.056 amu) This was greater than expected for this

instrument when used for internal comparison of two

ions The theoretical differences for Q47N or T45S

(14.016 amu) were within the expected error (15 p.p.m)

MS⁄ MS analysis (Fig 2 and Table 1) confirmed the

T45S difference Peptide fragmentation at the C–N

peptide bond gives b ions from the N-terminus and y

ions from the C-terminus (Table 1) Cleavage at the

C–C bond provides a ions from the N-terminus All

ions from the N-terminus were 14 amu lower for the

m⁄ z ¼ 865.465 peptide, whereas all ions from the

C-terminus as well internal ions were identical for the two peptides Detection of the same ions with similar relative intensities (Fig 2) established the near identity

Table 1 MS ⁄ MS analysis of relevant ApoC1 peptides identified by BIOANALYST software To conserve space, ions are rounded to 1 decimal place The m ⁄ z values were accurate to three places ND, not determined.

(N-terminal ions)

observed ⁄ theoretical

(C-terminal ions) observed ⁄ theoretical

(N-terminal ions) observed ⁄ theoretical

(C-terminal ions) observed ⁄ theoretical

Internal ions common to both peptides

(Immonium of Q) 101.1 ⁄ 101.1 (Internal QK) 257.2 ⁄ 257.2

(Immonium of F) 120.1 ⁄ 120.1 (Internal QK-H 2 O) 239.2 ⁄ 239.2

(K rearrangement) 129.1 ⁄ 129.1 (Internal QK-NH3) 240.1 ⁄ 240.1

(Immonium of K-NH3) 84.08 ⁄ 84.08 –

b 5 257

101

239 240

A

300

0 10 20 30

879

129

a 2

b 6

b 5 -H20

b 4

b 3

b 2

y 2

y 4

m/z

0 10 20

30

b 5

b 6

b 4

b 3

b 2

a 2 129

257 b

5 H20

865

y 2 y4

101

239 240

B

70 120

Fig 2 MS ⁄ MS spectra of peptides of m ⁄ z ¼ 879.468 (top panel) and 865.465 (bottom panel) The a, b and y ions are labeled and presented in Table 1 Internal ions are labeled by m ⁄ z values roun-ded to the nearest mass unit.

of the two peptides, except for the N-terminus The

T45S mutation requires a single base change (A267T,

accession number X00570)

Altered distribution of the S45 variant in VLDLs

compared with HDLs

Ultracentrifugation of plasma partially separates

lipo-protein classes VLDLs float to the top (fraction 1,

Fig 3), while HDLs sediment near the middle and

bot-tom of the tube Earlier studies showed that peak

intensity ratios provide an estimate of the relative

pro-tein ratios in different samples [19] As expected from

known distributions of the apolipoproteins, the relative

abundance determined by peak intensity ratios of

ApoC2 (m⁄ z 8915) and ApoC3 (sum of m ⁄ z ¼ 8765,

9422, 9642 and 9713) to ApoC1 was greatest in VLDL

fractions and declined in HDL fractions (Fig 3F)

With the same approach, the S45 variant of ApoC1

partitioned more to the VLDL fraction than the T45

variant (compare Fig 3A and 3B with 3C and 3D) The mean ± SD from triplicate runs for three frac-tions was determined (Fig 3E) A single analysis of every fraction showed that the primary change occurred between fractions 3 and 8 (not shown), as expected for the transition from VLDL to HDL and other classes of lipoproteins In the experiment shown, the S45 variant of ApoC1 showed 1.6-fold greater abundance than the T45 variant in VLDLs (fraction 1) compared with HDLs (fractions 8 and 16, Fig 3E) This enrichment of the low-mass component in VLDLs was observed in all eight people whose plasma was analyzed by this method (average difference¼ 1.5-fold) Once again, single profiles taken of each fraction showed that the majority of change occurred between fractions 3 and 8

Selective incorporation of other protein isoforms in VLDLs compared with HDLs was not observed For example, the ratios of truncated to full-length ApoC1 were constant across the ultracentrifuge fractions for both the S45 and T45 variants (open symbols, Fig 3E)

as were the ratios of four isoforms of ApoC3 (open symbols, Fig 3F) This suggests that the T45S change had altered the lipid-interaction site in a manner that changed lipid-binding specificity

m/z

500

1000 2000

2000

6600 6640

6400 6440

6420

6434

6618 6632

VLDL

HDL

Fraction No

0 5 10 15

F

0

0.4

0.8

1.0

1.4

E

Fig 3 Differential distribution of apolipoproteins plus isoforms and variants in VLDLs compared with HDLs (A, C) Relevant sections of profiles (2000 laser shots, attenuation 44) showing truncated forms

of ApoC1 (m ⁄ z ¼ 6434 and 6420) in fractions 1 and 9 of the ultra-centrifuge tube, respectively Fraction 1 represents VLDLs at the top of the tube (B, D) Relevant sections of profiles showing full-length forms of ApoC1 (m ⁄ z ¼ 6632 and 6618) in fractions 1 and 9

of the ultracentrifuge tube, respectively (E) Relative peak intensity ratios for the T45:S45 variants of the full-length (m ⁄ z ¼ 6618 ⁄ 6632,

r ) and truncated (m ⁄ z ¼ 6420 ⁄ 6434, n) forms of these proteins The peak ratio in fraction 1 was assigned a value of 1.0, and the ratios in subsequent fractions are expressed relative to that value Also shown are peak ratios for the full-length to truncated forms of the T45 variant (m ⁄ z ¼ 6632 ⁄ 6434, e) and the S45 variant (m ⁄ z ¼

6618 ⁄ 6420, h) (F) Peak ratios of lipoprotein isoforms The ratio of peak intensities for ApoC2 (m ⁄ z ¼ 8915, n) to the sum of peaks of ApoC1 was determined for fraction 1 and assigned a value of 1.0 The peak ratios in subsequent fractions are expressed relative to that value Also shown are the relative ratios of the sum of peaks from ApoC3 to the sum of peak intensities from ApoC1 (r) and the ratios of several isoforms of ApoC3 (m ⁄ z ¼ 8765 : 9713, des-glycoApoC3-0 ⁄ ApoC3-2, n; m ⁄ z ¼ 9422 : 9713, ApoC3-1 ⁄ ApoC3-2,

e ; m ⁄ z ¼ 9642 : 9713, C-terminal truncated ApoC3-2 ⁄ ApoC3-2, X; and m ⁄ z ¼ 9932 : 9713, an unidentified form of ApoC3 (22) ⁄ ApoC3-2, s) Error bars represent the standard deviation of three measurements For clarity, only one set of error bars are shown for the ApoC3 variants The mean coefficient of variation for the experimental data points for the ApoC3 ratios was 7%.

Increased susceptibility to N-terminal truncation

in vivo and in vitro

In the plasma, peak intensity ratios suggested that the

S45 protein was more highly truncated than the T45

protein (Fig 1) In fact, T45 occurs midway in an

am-phipathic helix that participates in lipid contact [22]

(Fig 4) The S45 variant would have one fewer methyl

groups at the lipid interface, giving a theoretical

differ-ence in free energy of lipid binding of +0.68 kcalÆmol)1

[23] and a threefold change in binding constant at

37C In agreement with such a difference, degradation

by dipeptidylpeptidase IV (DPPase) in vitro occurred

approximately 3 times faster for the S45 than the T45

variant (Fig 5) Lower-affinity lipid contact of the S45

protein may have made this protein more susceptible to

N-terminal truncation in vitro as well as in vivo

Discussion

This study used MS profile analysis to detect an

altered protein pattern in a subgroup of individuals

with American Indian and Mexican ancestry We have

not observed this pattern in over 1000 persons of other

ethnic backgrounds MS fragmentation of a novel

pep-tide from one individual indicated a T45S variant of

ApoC1 To our knowledge, this is the first example of

a structural polymorphism of ApoC1 that has been

found Circumstantial evidence such as mass difference

from the common protein form and an enhanced level

of N-terminal truncation suggested that all persons

who displayed this pattern had the same structural

modification Further work is needed to confirm this

prediction

Several lines of evidence suggest that S45 ApoC1 differed functionally from the T45 protein First of all, peak intensities in the profile suggest that the S45 protein was more highly processed by N-terminal truncation than the T45 protein Use of peak inten-sity ratios to estimate relative protein abundance depends on equal crystallization of the protein in the matrix and equal ionization of the proteins in the mass spectrometer This assumption appears quite good for nearly identical structures [19] such as the proteins of a polymorphism pair (see also TTr, Fig 1C) Other quantitative evaluations presented in this study were even less dependent on identical prop-erties For example, the method used to estimate the rates of digestion by DPPase and the different distri-butions of the S45 variant among lipoprotein classes used comparison of peak ratios in different samples The conclusions from these experiments were not dependent on identical ionization of the two peptides but only on identical relative ionization of the two species in different samples

Variant proteins with identical function are synthes-ized and utilsynthes-ized at identical rates and should be pre-sent at equal concentrations in a sample If the variants have nearly identical chemical properties, they should give peaks of identical intensity in the mass spectrometer Indeed, most polymorphisms observed in our studies have presented double peaks of nearly

T45-Methyl

Fig 4 Molecular model of ApoC1 Structure 1 of the 35–53

pep-tides of ApoC1 in complex with SDS micelles [22] is depicted in

chains projecting upward in cpk color and the N-terminus on the

right Basic residues are in blue, and acidic residues in red The

methyl group of T45 is identified.

-0.4

-0.8

-1.2

0 100

Time, min

Fig 5 First-order decay plots for degradation of ApoC1 by hog kid-ney DPPase Results are for the common (m ⁄ z ¼ 6632, r, k ¼ )0.0022) and low-mass (m ⁄ z ¼ 6618, m, k ¼ )0.0069) variants of ApoC1 MS settings were as described in the legend to Fig 3 Means ± SD from three experiments are shown.

identical intensity (TTr in Fig 1, example in ref [19]

and unpublished results) In contrast, functionally

dif-ferent proteins often give peaks of unequal intensity

because of unequal rates of biosynthesis, utilization, or

degradation Variants of TTr that are associated with

pathophysiology often show unequal peak intensities

[24,25] The results for ApoC1 suggest that unequal

abundance applied to the full-length, low-mass variant

of ApoC1 (m⁄ z ¼ 6618, Fig 1) which showed lower

intensity than the common variant (m⁄ z ¼ 6632) In

contrast, the truncated, low-mass variant was often

slightly more intense than the truncated, common

vari-ant These properties suggest that the S45 variant was

more susceptible to truncation in the plasma, a

conclu-sion supported by increased rates of degradation by

DPPase in vitro

The enzyme thought to be responsible for truncation

of ApoC1 in vivo is DPPase [20], an important

regula-tor of insulin production through its inactivation of

the incretins, hormones that enhance insulin

produc-tion [26] Reduced activity of DPPase occurs in

per-sons with diabetes [27], and reduced truncation of

ApoC1 was observed in a case of hyperlipidemia [20]

The physical properties of ApoC1 led others to suggest

that the N-terminus is responsible for its regulatory

interactions [28] ApoC1 in HDLs [7] and an

N-ter-minal 38-residue form of ApoC1 are able to inhibit

cholesterol ester transferase [6] Potential regulation of

several lipase enzymes [3–5] increases the question of a

role for truncation in protein–protein interactions If

truncation serves a biological function, it may enhance

the functional differences between the S45 and T45

variants of ApoC1

A functional importance of a methyl group side

chain at position 45 was also suggested by homology

alignment ApoC1 from six available species shows

either Ala or Thr at the comparable position (Table 2)

The Ala-Phe or Thr-Phe motif is common at two

locations of ApoC1 (Table 2), and this combination may provide unique properties for interaction with lipoproteins

Future studies are needed to determine whether the T45S variation is common to all people who dis-play this profile, the possible effects of the T45S variant on lipid metabolism, and the role of N-ter-minal truncation of ApoC1 in vivo The T45S variant may offer an excellent tool for future studies with models such as transgenic animals, as it provides a form of ApoC1 that is more susceptible to trunca-tion in vivo Studies related to these questrunca-tions are in progress

Experimental procedures

Materials

Alpha-cyano-4-hydroxycinnaminic acid, HPLC-grade tri-fluoroacetic acid, and hog kidney DPPase IV were from the Sigma Chemical Co (St Louis, MO, USA) Sinapinic acid was from Roche (Mannheim, Germany), and sequencing-grade Glu-C protease was from Roche, Inc (Indianapolis,

IN, USA) HPLC-grade acetonitrile was from Mallinckrodt Baker Inc (Paris, KY, USA)

Protein profile analysis

The procedure to obtain MALDI-TOF protein profiles of plasma has been described elsewhere [19] along with prop-erties and identification of the proteins in the profile The method applied a very consistent extraction method to plasma proteins using reverse-phase C4 column material (the C4 ZipTip from Millipore Inc., Bedford, MA, USA) This method is applied directly to diluted plasma or serum without removal of abundant proteins Abundant proteins such as albumin and immunoglobulins do not appear in the profile with this method Use of alternative matrices or other changes does allow detection of the abundant pro-teins However, the profile method provides a consistent spectrum of plasma proteins such as ApoC1 ( 40 lgÆmL)1 versus nearly 40 mgÆmL)1) with high precision Sinapinic acid was used as the matrix, and the sample was analyzed

in a Bruker Biflex III MALDI-TOF mass spectrometer operating in the linear mode Five hundred laser shots (attenuation of 39%) were collected The data was smoothed using software (Bruker Daltonics xtof version 5.1.1) provided with the Biflex III (Golay-Savitzky formula using 15 points), and background was subtracted The pro-files were analyzed by peak intensity ratios, a very accurate method for comparison of protein concentrations in differ-ent samples [19] When necessary, resolution was increased

by use of greater laser attenuation with accumulation of

1000 or 2000 laser shots

Table 2 Sequence homology of ApoC1 from different species.

From Swiss-Prot Data Bank (http://us.expasy.org/sprot)

Hydropho-bic residues are in bold, and residues homologous to position 45 of

human ApoC1 are in large type This is residue number 49 in dog,

mouse, rat, and tree shrew.

Protein identification

Plasma (1.1 mL) was centrifuged at 160 000 g for 4 h in a

Beckman table-top ultracentrifuge The solution was

aspir-ated from the top of the tube into 20 equal fractions

MALDI-TOF profile analysis was conducted on each

frac-tion For protein identification, the upper three fractions

were pooled, and 20 lL was applied to one channel of an

SDS⁄ polyacrylamide gel with standard ApoC1

(CalBio-chem, San Diego, CA, USA) in an adjacent channel

One-dimensional SDS⁄ PAGE was performed on a Bio-Rad

(Hercules, CA, USA) Protean II xi system using 12%

Tris⁄ Tricine gels (16 cm · 16 cm) The gel was stained with

Coomassie blue, the protein band corresponding to ApoC1

was excised, and the gel slices were subjected to in-gel

digestion with sequence-grade Glu-C protease The

diges-tion and extracdiges-tion of the peptides from the gel were

accomplished by standard procedures Peptide fingerprint

maps of the digested proteins were obtained in both the

Bruker Biflex III operating in the reflectron mode and in a

QSTAR o-MALDI mass spectrometer (Applied Biosystems

Inc., Bellarica, CA, USA) MS⁄ MS analysis was conducted

with the QSTAR o-MALDI mass spectrometer The TOF

region acceleration voltage was 4 kV, and the injection

pulse repletion was 4.9 kHz with a pulse time of 18 ls The

pulses were generated by a nitrogen laser at 337 nm with

an energy of 9 lJ and a repetition rate of 19 Hz Each

spectrum was the sum of 300 laser shots The tandem mass

spectral data was collected using a collision energy of 37

(unitless) External calibration was performed with human

angiotensin II (monoisotopic [MH+] m⁄ z 1046.5417;

Sigma) and adrenocorticotropin (ACTH) fragment 18–39

(monoisotopic [MH+] m⁄ z 2465.1989; Sigma) Spectra

were analyzed with bioanalyst software (Applied

Biosys-tems, Inc.) The software, set at a tolerance of 100 p.p.m.,

identified a, b, y and internal ions

Enzyme degradation

Plasma (5 lL) that displayed the double peak for ApoC1

was diluted to 140 lL with Tris⁄ HCl (0.05 m,

pH 8.0)⁄ 0.1 m NaCl buffer DPPase (10 lL of 1 UÆmL)1)

was added, and the mixture incubated at 37C Aliquots

(15 lL) were removed at 20-min intervals, acidified with

0.5 lL 10% trifluoroacetic acid, extracted with a C4

ZipTip by standard procedure [19], and analyzed by the

protein profile method The fraction of full-length ApoC1

was determined from the peak intensity of full-length

pro-tein divided by the sum of intensities for full-length and

truncated ApoC1 The rate constant for disappearance of

ApoC1 was determined from a first-order decay plot, and

standard deviation from triplicate experiments The

analy-sis was applied to both variants of ApoC1 in the same

sample, thereby eliminating the need for intersample

comparison

Research subjects

All research subjects and studies were conducted with approval by the institutional review board of the University

of Minnesota, and informed consent was obtained for pro-tein analysis of the samples to discover biomarkers Blood was obtained by venepuncture, anticoagulated with sodium citrate, and centrifuged to obtain plasma Samples were from individuals studied in conjunction with several pro-jects designed to detect biomarkers related to obesity, insu-lin resistance, diabetes, graft versus host disease, heart disease, sepsis and a number of other conditions Many were healthy individuals, representing extensions of pub-lished studies [19,29,30] The ethnic background of at least

1000 subjects was typical of the American Midwest with

 90% of European ancestry and  5% each of African and Asian ancestry An exception was the targeted analysis

of 228 persons with American Indian ancestry and 86 per-sons of Mexican ancestry These groups were self-identified

by ethnic background and confirmed by independent phe-notype evaluation The latter groups provided 44 examples

of the unusual profile shown in this study, which reports detailed properties of the proteins from one person with Mexican ancestry

Acknowledgements

This work was supported in part by the endowment to the Samuel Kirkwood professorship (GLN) RSK was supported by a National Hemophilia Clinical Fellow-ship Award KM is a Bush Medical Fellow (Bush Foundation) The mass spectrometry and parts of the gel electrophoresis were conducted with the assistance

of Mr Thomas Krick, Drs LeeAnn Higgins, Lorraine Anderson, Sudha Marimanikkuppam and Bruce Witthuhn of the Center for Mass Spectrometry and Proteomics at the University of Minnesota (Director GLN) We acknowledge the expert technical assistance

of Ms Julia Nguyen and other volunteers who assisted

in sample procurement

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