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

A functional polymorphism of apolipoprotein C1 detected by mass spectrometry Matthew S. Wroblewski1, Joshua T. Wilson-Grady1, Michael B. Martinez1, Raj S. Kasthuri2, Kenneth R. McMillan3, Cristina Flood-Urdangarin4 and 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

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.

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 in vivo. functions that may be important potential 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

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.

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residue of

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

A

9422

6632

TTr

each component, expected N-terminal 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 alkylation. 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.

8915

9713

13762

13881

13762

y t i s n e t n I

B

13792

6632

C

TTr

ApoC1

6618

13881

6420

13911

6434

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 is disulfide-linked to cysteine (m ⁄ z ¼ a form that 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 (cid:2) 13% of samples and may represent a common G6S variant [21].

m/z

is,

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 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

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.

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

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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.

865.465 peptide (SFQKVKE)

879.468 peptide (TFQKVKE)

(C-terminal ions) observed ⁄ theoretical

(N-terminal ions) observed ⁄ theoretical

(C-terminal ions) observed ⁄ theoretical

(N-terminal ions) observed ⁄ theoretical

– (y6)ND (y5)ND (y4)503.3 ⁄ 503.3 (y3)ND (y2)276.2 ⁄ 276.2 (y1)ND – –

– (y6)ND (y5)ND (y4)503.3 ⁄ 503.3 (y3)ND (y2)276.1 ⁄ 276.2 (y1)ND – –

(b1-H2O)ND (b1)ND (b2)235.1 ⁄ 235.1 (b3)363.2 ⁄ 363.2 (b4)491.3 ⁄ 491.3 (b5)590.3 ⁄ 590.3 (b6)718.4 ⁄ 718.4 (a2)207.1 ⁄ 207.1 (a5)562.3 ⁄ 562.3

(Internal QK) 257.2 ⁄ 257.2 (Internal QK-H2O) 239.2 ⁄ 239.2 (Internal QK-NH3) 240.1 ⁄ 240.1 –

(b1-H2O)84.0 ⁄ 84.0 (b1)ND (b2)249.1 ⁄ 249.1 (b3)377.2 ⁄ 377.2 (b4)505.3 ⁄ 505.3 (b5)604.4 ⁄ 604.4 (b6)732.5 ⁄ 732.4 (a2)221.1 ⁄ 221.1 (a5)576.4 ⁄ 576.4 Internal ions common to both peptides (Immonium of Q) 101.1 ⁄ 101.1 (Immonium of F) 120.1 ⁄ 120.1 (K rearrangement) 129.1 ⁄ 129.1 (Immonium of K-NH3) 84.08 ⁄ 84.08

b5

A

879

257

239 240

101

30

b4

the

a2

20

y4

b5-H20

b3

129

b2 y2

10

84

b6

861

0 30

865

b5

101 B

s t n u o c , y t i s n e t n I

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 truncated protein (residues 1–13, TPDVSSALDKLKE, theoretical mass ¼ 1402.7 amu; residues 3–13 (the truncated protein), 1204.7 amu; resi- dues 14–19, FGNTLE, 680.3 amu; residues 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).

129

239 240

20

a2

b2

257

b5 H20 -

70

b3

b4

10

120

y4

b6

represented

(14.003 amu, Table 1)

y2

0

100

200

300

400

500

600

700

800

m/z

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.

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

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 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

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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

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 difference ¼ 1.5- fold). Once again, single profiles taken of each fraction showed that the majority of change occurred between fractions 3 and 8.

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

6420

6632

A

B

2000

1000

6618

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.

VLDL

6434

y t i s n e t n I

C

D

2000

500

HDL

6600

6640

6400

6440

m/z

E

1.4

1.0

0.8

l

o i t a r k a e P e v i t a e R

unidentified

an

0.4

F

0

0

10

15

5 Fraction No.

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 isoforms of ApoC3 (m ⁄ z ¼ 8765 : 9713, des- the ratios of several 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, 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%.

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Increased susceptibility to N-terminal truncation in vivo and in vitro

-0.4

i

) n e t o r P h t g n e l l l

-0.8

u f n o i t c a r F ( n I

-1.2

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 37 (cid:2)C. 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.

0

100

Time, min

Discussion

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.

Several

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.

T45-Methyl

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 spectrometer. This assumption appears quite mass 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. from these experiments were not The conclusions dependent on identical ionization of the two peptides but only on identical relative ionization of the two species in different samples.

in a sample.

If

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

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 RASMOL. The helix is in a space-filled model with hydrophobic side 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.

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

the possible effects of

Future studies are needed to determine whether the T45S variation is common to all people who dis- play this profile, 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 questions are in progress.

Experimental procedures

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.

Materials

Protein profile analysis

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

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.

Sequence

Species

Human S45 Human Baboon Dog Mouse Rat Tree shrew

ELSAKMREWFSESFQKVKEKLKIDS ELSAKMREWFSETFQKVKEKLKIDS EFPAKTRDWFSETFRKVKEKLKINS DIPAKTRNWFSEAFKKVKEHLKTAFS EILTKTRAWFSEAFGKVKEKLKTTFS EIMIKTRNWFSETLNKMKEKLKTTFA DLPAKTRNWFTETFGKVRDTFKATFS

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).

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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 ((cid:2) 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.

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Protein identification

Research subjects

Acknowledgements

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 (cid:2) 90% of European ancestry and (cid:2) 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.

(monoisotopic [MH+] m ⁄ z

Enzyme degradation

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.

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 extraction 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 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.

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