Identification and Characterization of Apelin Peptides in Bovine

ARTICLE pubs.acs.org/jpr

Identification and Characterization of Apelin Peptides in Bovine Colostrum and Milk by Liquid Chromatography Mass Spectrometry Cedric Mesmin,† Franc-ois Fenaille,† Franc-ois Becher,† Jean-Claude Tabet,‡ and Eric Ezan*,† † ‡

CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, France CNRS-UPMC, CSOB, UMR 7613, France ABSTRACT: Apelin peptides were recently identified as endogenous ligands of the APJ receptor. It has been hypothesized that these peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development. As apelin peptides may have different effects on the APJ receptor as a function of their size, knowledge of their exact structure in early milk is essential to clarify their action in gastrointestinal tract development. Bovine colostrum is thought to contain high concentrations of a wide diversity of apelin peptides, but none of them have yet been rigorously characterized. To identify and monitor apelin peptides in bovine colostrum, we developed a cation exchange extraction step followed by untargeted liquid chromatography coupled to high resolution and high mass accuracy mass spectrometry (LTQOrbitrap). Using this approach, we characterized 46 endogenous apelin peptides in bovine colostrum, which varied in relative abundance from one colostrum to another. Mature as well as commercial milk samples were also studied. Taken together, our data demonstrate that the multiplicity and variability of apelin peptides are biologically relevant and change during milk maturation to reach a more constant composition in mature milk. KEYWORDS: apelin, mass spectrometry, bovine colostrum, bovine milk

’ INTRODUCTION Apelin peptides were recently identified as endogenous ligands of the APJ receptor, a G-protein-coupled receptor.1 These peptides are derived from a 55-amino-acid proapelin that contains in its C-terminal part 23 amino acids fully conserved between human and bovine species.2 Considering its numerous basic residues, proapelin is thought to be processed into bioactive peptides bearing from 12 (apelin-12) to 36 (apelin-36) amino acids in the C-terminal part (Figure 1A).1,3 Apelin peptides and their receptor are found in the heart, lung, brain, kidney, adipose tissue, vascular epithelium, gastrointestinal tract and mammary gland.4 Particular attention has been paid to the relevance of apelin peptides as a heart failure biomarker,5 8 and an ever growing number of studies have demonstrated their involvement in numerous physiological activities (heart contractility and blood pressure,9 appetite and drinking behavior,10 glucose metabolism11, etc.) and diseases (acute myocardial infarction,12 diabetes,13 stable angina14, etc.). In particular, the apelin-APJ system has emerged as an actor in the physiology of the mammalian gastrointestinal tract.15 19 Apelin-13 stimulates gastric cell proliferation in vitro,16 and the subcutaneous administration of synthetic apelin peptides to mice with colitis stimulates colonic epithelial cell proliferation, suggesting an involvement in gastrointestinal tract maintenance.18 During rat adulthood, gastrointestinal apelin seems to be mainly produced in the glandular epithelium of the stomach and might act on APJ receptors localized in the duodenum and colon.15,16 In the developing rat, gastric apelin-producing cells are absent until 20 days of age,16 whereas APJ r 2011 American Chemical Society

receptors are abundant in the duodenum and colon.15 Considering the secretion of apelin peptides by the mammary gland into colostrum and early milk,3,20 these results suggest that apelin peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development.15 As apelin peptides may have greater or lesser effects on the APJ receptor as a function of their size,4 knowledge of the nature of the apelin peptides released in early milk is essential to clarify their action in gastrointestinal tract development. Specific bio- and immunoassays show that apelin peptide concentrations in human, rat and bovine early milk are 5, 100 and up to 200 ng/mL, respectively.20,21 As bovine milk contains high apelin concentrations, is easily available and is of great nutritional significance to humans, its apelin content has been extensively investigated. Comparison of the gel filtration retention times of endogenous fragments and synthetic peptides, combined with subsequent specific receptor-binding bioassay detection, has suggested the presence of a wide diversity of bioactive apelin peptides in bovine colostrum and milk.3,20 However, none of these peptides have yet been strictly characterized in this matrix using common physical chemical techniques such as Edman sequencing and mass spectrometry. Several studies have proven the potential of liquid chromatography coupled to mass spectrometry (LC MS) in identification and characterization of bioactive peptides and proteins in complex Received: August 2, 2011 Published: September 23, 2011 5222

dx.doi.org/10.1021/pr200725x | J. Proteome Res. 2011, 10, 5222–5231

Journal of Proteome Research

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Figure 1. FTMS analysis for apelin peptide identification in cow 1 colostrum. (A) Sequences of human and bovine apelin-55 (proapelin), (B) FTMS total ion chromatogram, (C) deconvoluted mean FTMS spectrum over the retention time range 3 8 min. Synthetic stable isotope labeled human apelin-36 (lab. hum. ap-36) was spiked at 50 ng/mL before extraction.

matrices such as milk and colostrum.22 26 To determine which apelin peptides are released in bovine colostrum and milk, we have developed an untargeted approach based on an ion exchange extraction process previously developed for the extraction of apelin peptides from human plasma,27 followed by high resolution and high mass accuracy mass spectrometric analysis using an LTQ-Orbitrap instrument.

’ EXPERIMENTAL SECTION Chemicals and Reagents

Human apelin-36 and stable isotope labeled human apelin36 were synthesized by Bachem (Weil am Rhein, Germany) and used for extraction normalization. Stable isotope labeled proline residues ([13C5;15N]Pro) were incorporated into the human apelin-36 sequence at positions 13, 26, and 33. The final molecular mass shift relative to unlabeled peptides was 18 Da. Formic acid (HPLC grade) and phosphate saline buffer saline were from Sigma (Saint Quentin Fallavier, France). HPLC-grade acetonitrile (ACN) was from SDS (Peypin, France). Sample Preparation for the Analysis of Bovine Colostrum and Milk Samples by LC MS

Bovine colostrums from 10 healthy cows, milk from 3 healthy cows, and milk samples pooled from livestock cows were obtained

from a local farm (La ferme de Viltain, Jouy-en-Josas, France). Commercial pasteurized and sterilized bovine milk samples were purchased in local supermarkets. After collection, bovine milk samples were stored at 4 °C during transportation and centrifuged at 4 °C for 5 min at 2000 g. The lipid supernatant was then removed, and the partially defatted milk sample was stored at 80 °C until analysis. To study the stability of the apelin peptides under these conditions, each sample was divided into 2 aliquots. One was immediately supplemented with an antiprotease cocktail (80 μL for 4 mL of milk) of aprotinin (0.5 mg/mL), leupeptin (0.5 mg/mL), pepstatin A (0.5 mg/mL), benzamidine (31 mg/mL) and phenylmethylsulfonyl fluoride (17.5 mg/mL), and immediately frozen at 80 °C until analysis. The other aliquot was incubated for 24 h at 4 °C. Prior to LC MS analysis, milk samples were defrosted on ice. Magnetic carboxylic acid beads (Dynabeads MyOne carboxylic acid, Invitrogen, Carlsbad, CA) were used for apelin peptide extraction. Bead suspension (240 μL) was added to 4000 μL of milk just after spiking labeled human apelin-36 to 50 ng/mL and the mixture was incubated for one minute on ice. Milk was removed, and magnetic beads were resuspended in 1 mL of phosphate buffer saline and then washed twice with 400 μL of phosphate buffer saline, once with 100 μL of water, and finally eluted with 50 μL of an acidic solution (water/ acetonitrile, 90:10 v/v, containing 1% formic acid). The resulting 5223

dx.doi.org/10.1021/pr200725x |J. Proteome Res. 2011, 10, 5222–5231


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Table 1. Apelin peptides identified in bovine colostrum monoisotopic

monoisotopic

retention time

m (Da)

z

theoretical m/z

experimental m/z

(min)

ap-55

6281.3092

9

698.9305

698.9299

7.93

0.9

FTMS, FTMS2, FTMS3, ITMS3

7/10

ap-53

6127.2350

9

681.8112

681.8104

7.42

1.1

FTMS, FTMS2, ITMS2

9/10

ap-53/F

5980.1666

9

665.4702

665.4746

6.91

6.6

FTMS, FTMS2

8/10

ap-52

6014.1510

9

669.2463

669.2475

7.16

1.8

FTMS, FTMS2

ap-52/F

5867.0825

9

652.9053

652.9092

6.66

5.9

FTMS

ap-51

5901.0669

9

656.6814

656.684

6.91

4.0

FTMS

3/10

ap-51/F ap-p51

5753.9985 5884.0403

9 9

640.3404 654.7895

640.3429 654.7919

6.58 7.16

3.8 3.6

FTMS, FTMS2 FTMS

6/10 4/10

ap-p51/F

5736.9719

9

638.4486

638.4509

6.74

3.6

FTMS

7/10

ap-37

4339.3627

8

543.4276

543.4273

6.1

0.6

FTMS

1/10

ap-36

4176.2993

8

523.0447

523.0454

5.88

1.4

FTMS, FTMS2, ITMS3

10/10

ap-36/F

4029.2309

8

504.6611

504.6618

5.09

1.3

FTMS, FTMS2

10/10

ap-36/PF

3932.1781

8

492.5295

492.5268

4.29

5.6

FTMS

10/10

ap-35

4063.2153

8

508.9092

508.9111

5.53

3.8

FTMS, FTMS2

10/10

ap-35/F ap-35/PF

3916.1468 3819.0941

8 8

490.5256 478.3940

490.5275 478.3950

4.65 4.39

3.8 2.0

FTMS FTMS, FTMS2

10/10 9/10 10/10

name

Δ ppm

identification dataa

occurrence

5/10 10/10

ap-35/MPF

3688.0536

8

462.0140

462.0157

3.66

3.7

FTMS, FTMS2

ap-31

3582.9344

7

512.8550

512.8551

5.53

0.1

FTMS, FTMS2

8/10

ap-31/F

3435.8660

7

491.8453

491.8452

4.47

0.2

FTMS, FTMS2

10/10

ap-28

3272.7591

6

546.4671

546.4688

5.71

3.1

FTMS, FTMS2

ap-28/F

3125.6906

6

521.9557

521.9571

4.65

2.7

FTMS

10/10

ap-28/PF

3028.6379

6

505.7803

505.7812

4.39

1.9

FTMS

10/10

ap-27 ap-27/F

3185.7270 3038.6586

6 6

531.9618 507.4504

531.9631 507.4514

5.62 4.56

2.5 2.0

FTMS FTMS

5/10 9/10

ap-27/PF

2941.6059

6

491.2749

491.2781

4.29

6.5

FTMS

6/10

ap-25

3031.6528

6

506.2827

506.2849

5.44

4.3

FTMS

1/10

ap-25/F

2884.5844

6

481.7713

481.7731

4.25

3.6

FTMS

1/10

ap-25/PF

2787.5316

6

465.5959

465.5971

4.41

2.6

FTMS

1/10

9/10

ap-24/F

2827.5629

6

472.2678

472.2705

4.83

5.8

FTMS

2/10

ap-24/PF

2730.5102

6

456.0923

456.0925

3.57

0.4

FTMS

6/10

ap-23 ap-23/F

2877.5786 2730.5102

6 6

480.6037 456.0923

480.6050 456.0925

5.18 3.57

2.7 0.4

FTMS FTMS

1/10 5/10

ap-23/PF

2633.4574

6

439.9168

439.9177

3.25

1.9

FTMS

2/10

ap-22

2691.4993

6

449.5905

449.5891

5.07

3.1

FTMS

2/10

ap-19

2449.3978

6

409.2402

409.2393

4.5

2.3

FTMS

3/10

ap-18

2293.2966

5

459.6666

459.6664

4.8

0.4

FTMS, FTMS2

7/10

ap-17

2137.1955

5

428.4464

428.4487

5.09

5.4

FTMS, FTMS2

4/10

ap-17/F

1990.1271

5

399.0327

399.0323

5.27

1.0

FTMS, ITMS2

10/10

ap-16 ap-15

2009.1006 1862.0322

5 5

402.8274 373.4137

402.8284 373.4146

5.45 5

2.5 2.4

FTMS, FTMS2 FTMS, FTMS2

4/10 7/10

ap-15/F

1714.9637

5

344.0000

344.0008

2.36

2.3

FTMS

1/10

ap-14

1705.9310

4

427.4900

427.4905

5.45

1.1

FTMS, FTMS2

5/10

ap-13

1549.8299

4

388.4648

388.465

6.06

0.6

FTMS

2/10

ap-13/F

1402.7615

4

351.6977

351.6977

3.47

0.1

FTMS

3/10

ap-12

1421.7714

4

356.4501

356.4513

6.06

3.3

FTMS, FTMS2

7/10

ap-12/F

1274.7029

4

319.6830

319.6830

3.1

0.0

FTMS

8/10

a

FTMS2 and/or ITMS2 identification data were not available for molecular species with low-intensity signals because of uninterpretable noisy MS/MS spectra.

mixture was transferred to a polypropylene vial and 40 μL was injected into the chromatographic system. For evaluation of the extraction yield, 4 mL of a pool of 10 bovine colostrums was spiked with 200 ng of synthetic human apelin-36.

Extraction was then performed as previously described and 200 ng of labeled human apelin-36 was added to 50 μL of elution solution. Extraction rate was then calculated as the ratio between the unlabeled and labeled human apelin-36 chromatographic peak areas. 5224


Journal of Proteome Research Chromatographic and Mass Spectrometric Conditions

LC MS experiments were performed using an Accela HPLC system coupled to an LTQ-Orbitrap mass spectrometer, both from Thermo Scientific (San Jose, CA). Chromatographic separation was performed on a Polaris C18-A column (150 2 mm i.d., 3 μm particle size) from Varian (Palo Alto, CA). The column was eluted at a flow rate of 0.2 mL/min using water as mobile phase A and acetonitrile as mobile phase B, both containing 0.1% formic acid. A linear gradient from 5 to 35% B in 10 min was performed. From 10 to 40 min, a linear gradient was set to reach 100% B and the column was washed for 10 min at this buffer composition. The equilibration time before the next analysis was set at 10 min, so that the time between two experiment cycles was 60 min. Column temperature was maintained at 40 °C. The column effluent was directly introduced into the electrospray source of the LTQ-Orbitrap mass spectrometer. Analyses were performed in the positive ion mode, and sequential MSn experiments, based on collision-induced dissociation (CID) with helium by radial resonant excitation, were performed using a classic top 3 data-dependent acquisition method. The electrospray voltage, capillary voltage and tube lens voltage were set at 5 kV, 35 and 80 V respectively. An in-source potential of 5 V was used. The sheath, ion sweep and auxiliary gas flows (nitrogen) were optimized at 30, 25, and 15 (arbitrary units), while the capillary temperature was set at 275 °C. For MS experiments, the resolution and maximal injection time were set at 30 000 (at m/z 400) and 1000 ms, respectively. Ion population in the Orbitrap analyzer was held at 3 106 through the use of automatic gain control. During sequential MS/MS experiments, multiply protonated molecular species bearing more than 2 protons were isolated in the LTQ analyzer with an isolation width of 3 m/z, and fragmented by low-energy CID. Automatic gain control of the LTQ, automatic gain control of the Orbitrap, normalized collision energy in LTQ, activation time and activation Q (position of the precursor ion on the qz scale) were set at 3 106 ions, 3 106 ions, 35 (arbitrary units), 30 ms and qz = 0.250 (dimensionless stability parameter), respectively. The resulting product ions were analyzed either directly by radial ejection in LTQ or by ion transfer by axial ejection through C-trap to the Orbitrap analyzer.

’ RESULTS AND DISCUSSION Apelin Peptide Extraction from Bovine Colostrum and Milk Samples

Like other biological fluids, bovine milk and colostrum are complex matrices that contain a wide and variable range of proteins. As an example, mature milk contains 3.3% protein, about 80% of which consists of caseins, the remaining 20% being whey proteins (serum albumin, β-lactoglobulin, α-lactalbumin and other lowabundance species).25 Colostrum has a distinct composition in IgG, IgA and IgM immunoglobulins, which are present at particularly high levels.23 With a global concentration of about 200 ng/mL,20 apelin peptides represent less than 10 4 % of the total protein content of bovine colostrum.28 Thus, analysis of such low-abundance species requires a specific enrichment process prior to LC MS analysis. After partial delipidation, apelin peptides were extracted from bovine colostrum using micrometric magnetic cation exchange beads, which we have previously used for the efficient extraction of apelin peptides from human plasma.27 The 23 amino acids of the C-terminal part of the proapelin (apelin-55) are shared by human and cattle and contain most of the basic lysine and arginine

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residues (Figure 1A). We therefore assumed that the behavior of bovine apelin peptides during the cation exchange extraction process could be efficiently mimicked and normalized by synthetic stable isotope labeled human apelin-36. Bovine and synthetic stable isotope labeled human apelin-36 differ in molecular mass by 35.049 Da, due to slightly different amino acid compositions and the incorporation of labeled proline residues. It should be pointed out that endogenous molecular species exhibiting the same monoisotopic mass than the stable isotope labeled human apelin-36 were not detected in bovine colostrum and milk. Thus, the chromatographic peak intensity of labeled human apelin-36 spiked at 50 ng/mL in each sample was used for data normalization. Moreover, the extraction rate for synthetic human apelin-36 was evaluated in a pool of 10 bovine colostrums as 40 ( 6% (n = 3) (data not shown). This extraction yield is lower than the 71% previously obtained for the same method in human plasma.27 The difference is not unexpected since human plasma and bovine colostrum are of different viscosity and fat composition. The LC MS method detection limit for synthetic human apelin-36 was also estimated as ∼0.5 ng/mL on the basis of a signal-to-noise ratio higher than 3 (data not shown). Identification of Apelin Peptides in Bovine Colostrum

The extraction method previously described was applied to 10 individual bovine colostrums and the eluates were analyzed using an LC ESI LTQ-Orbitrap working in the data-dependent acquisition mode. A typical FTMS (R = 30 000 at m/z 400) total ionic current chromatogram obtained for the colostrum of cow 1 is shown in Figure 1B. Most of the extracted species were eluted during the first 20 min of the analytical run, and the most intense chromatographic peaks had a retention time between 8 and 13 min. A Sequest analysis of the data obtained revealed that the most abundant peaks at 8.37, 8.74, 9.38, 9.80, and 11.19 min correspond to rather basic peptides from αS2, αS1, αS2, αS1 and αS1 casein, respectively (data not shown). However, it should be noticed that no apelin peptides were identified when corresponding MS and MS/MS spectra were searched using the Sequest algorithm (with no enzyme specificity) against a restricted database consisting of bovine protein sequences. Given the global apelin peptide concentration of around 200 ng/mL previously reported in bovine colostrum20 and the ng/mL sensitivity of our method, apelin peptides should be readily detected. We therefore performed manual spectral analysis of each chromatogram to look for theoretical apelin peptide masses. The deconvoluted spectrum obtained for the colostrum of cow 1 over 3 to 8 min (time range bracketing the retention time of the stable isotope labeled human apelin-36) is presented in Figure 1C. More than 20 molecular masses matching potential apelin peptides masses were identified with a mass accuracy in the 1 8 ppm range (Figure 1C). Potential apelin peptides detected in this colostrum range from apelin-12 to apelin-55 (proapelin) (Figure 1C). Applying the same approach to 10 different colostrums, 46 potential apelin peptides were detected (Table 1). To confirm the identification of the apelin peptides potentially detected, sequential MSn experiments under resonant CID excitation conditions were performed. As an example, apelin55 eluting at 7.93 min (Figure 2A) exhibited a typical ESI-FT mass spectrum where multiprotonated peptides bearing 6 to 11 charges were detected, while the prominent ions bore 9 and 10 protons (Figure 2B). Such high charge numbers are in good agreement with the numerous basic gas phase residues (lysine, histidine and arginine) in the apelin-55 sequence (Figure 1A). 5225



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Figure 2. Apelin-55 mass spectrometric characterization in colostrum of cow 1. (A) FTMS extracted ion current chromatogram of the most intense natural isotope of [apelin-55 + 9H+]9+, (B) FTMS mass spectrum obtained at retention time 7.93 min, (C) FTMS natural isotopic distribution of [apelin-55 + 9H+]9+, (D) sequential MS2 product ion spectrum obtained by radial excitation of [apelin-55 + 9H+]9+ (m/z 699.37) by CID into LTQ and detected using FTMS, and (E) sequential CID-MS3 experiment on the apelin-55 y528+ product ion (m/z 753.15) by radial excitation/detection into LTQ.

The natural isotopic distribution of the [apelin-55 + 9H]9+ ion revealed a monoisotopic m/z of 698.9299 with measurement accuracy better than 1 ppm (Figure 2C). This nona-protonated molecular species was next selected as precursor ion (within its total isotopic cluster) to perform sequential MS2 experiments in the LTQ before analysis of the resulting product ions in the Orbitrap mass analyzer. Product ions assignments have been achieved thanks to an average mass accuracy better than 2.5 ppm. Most of the product ions detected corresponds to octa- and hepta-charged y-type ions generated by loss of N-terminal amino acids (Figure 2D). These yj product ions (with j = 45 to j = 53, in addition to the y417+, and y427+ ions), along with the detection of b31+, b41+, and b51+ ions, allow unambiguous sequencing of the 10 first amino acids of the N-terminal part of the peptide (Figure 2D, Figure 1A). Moreover, the presence of the y143+ ion gives reliable information regarding the C-terminal sequence of the apelin-55 (Figure 2D, Figure 1A). Finally, the y21+ product ion detected corresponds to the cleavage before the proline residue of the C-terminal part and has been observed in synthetic human apelin peptide fragmentation.27 Curiously, the other proline residues at the 4, 11, 24, 26, and 33 positions (from C-terminus) do not induce dissociations or enhance cleavage rate constants. Likely, the presence of numerous basic histidine,

lysine, and arginine residues which keep the charges remote could be at the origin of these dissociation hindrances. However, the generation of prominent y-type ions was expected since the C-terminal part of the apelin-55 contains numerous basic residues (Figure 1A). To go further in the apelin-55 sequence analysis, sequential MS3 experiments on the most abundant y528+ product ion of the MS2 spectra were done (selection, radial resonant CID excitation and product ion detection into LTQ, Figure 2E). The fragmentation pattern obtained was similar to that of MS2, with a series of y-type ions generated by loss of N-terminal residues, confirming the N-terminal sequence of the apelin-55 previously established (Figure 2E). Thus, unambiguous identification and characterization of the apelin-55 peptide can be achieved, through the very high resolution/high mass accuracy of the Orbitrap analyzer in both MS and MS2. This approach was used to confirm the structures of all of the potential apelin peptides (Table 1). Interestingly, for the 10 colostrum samples studied, we were unable to detect any signal for apelin-50 to apelin-38 (Table 1). Two groups clearly appear, the first one comprising long forms of apelin peptides (from apelin-55 to apelin-51) and the other one consisting of shorter forms (from apelin-37 to apelin-12) (Table 1, Figure 1C). One possible explanation for this observation is cleavage 5226



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Figure 3. Apelin peptide stability in bovine colostrum. Colostrum was collected either with or without antiprotease cocktail. Synthetic stable isotopelabeled human apelin-36 was spiked at 50 ng/mL just before extraction and used for normalization of all other signals (relative intensity). (A) Colostrum aliquots were supplemented with the antiprotease cocktail and analyzed directly. (B) Untreated aliquots of colostrum were incubated at 4 °C for 24 h before analysis.

of apelin-55 by serine proteases after arginine/lysine residues in positions 38, 32, 29, 19, 18, 17, 15, and 14 of the C-terminal side, resulting in the generation of apelin-37, -31, -28, -18, -17, -16, -14 and -13, respectively. Subsequent nonspecific aminopeptidase cleavages of these latter fragments might finally produce the wide diversity of apelin peptides observed. A hypothetical degradation process such as this has already been suggested for apelin-5520 and is supported by the presence of peptidases in bovine colostrum (serine-endopeptidase, metalloendopeptidase, aminopeptidase).23 In addition to apelin peptides with an intact C-terminal sequence, apelin fragments lacking up to 3 amino acids from the C-terminal part have been identified (e.g., apelin-53/F, apelin35/PF and apelin-35/MPF lacking the C-terminal phenylalanine, proline-phenylalanine and methionine-proline-phenylalanine, respectively, cf. Figure 1A) (Table 1, Figure 1C). Apelin peptides are known substrates of the carboxypeptidase Angiotensin-converting enzyme 2 (ACE2)29 which might be involved in the C-terminal degradation observed, along with nonspecific carboxypeptidases. This is the first time that the apelin peptide family has been so extensively described, and the molecular diversity reported here is in good agreement with previous observations based on gel filtration and subsequent bioassay detection, suggesting huge structural heterogeneity.20

Stability of Apelin Peptides in Bovine Colostrum

The presence of proteases and truncated apelin peptides in bovine colostrum raises the question whether the latter are produced by artifactual degradation occurring in vitro during sampling and analysis. Such in vitro instability of human apelin12, -13, -p13, -17, and -36 has already been demonstrated in human plasma.27 A protease inhibitor cocktail along with sample processing at 4 °C has been successfully implemented to increase their stability.27 All the samples analyzed in this study were kept at 4 °C during transportation and processing (less than 24 h), and were stored at 80 °C until analysis. To investigate the stability of the apelin peptides under these conditions, three colostrums were collected either with or without antiprotease cocktail. Colostrum aliquots supplemented with the antiprotease cocktail were analyzed directly (Figure 3A), whereas untreated aliquots were incubated at 4 °C for 24 h before analysis (Figure 3B). Patterns obtained under both conditions were quite similar, with the presence of the same apelin peptides in comparable abundances. Forty apelin peptides were detected under the two conditions, and apelin-35/F exhibits the highest relative intensities in both profiles. Among the peptides detected, relative intensity variations of 31 species are below 40%. Such minor variations may be attributed to analytical variability since the MS analysis is not fully 5227



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Figure 4. Interindividual variability in apelin peptide composition in bovine colostrum.Colostrum samples from 10 individual cows were collected and analyzed by FTMS. Synthetic stable isotope-labeled human apelin-36 was spiked at 50 ng/mL before extraction and used for normalization of all other signals (relative intensity).

standardized by the use of stable isotope-labeled synthetic human apelin-36. Analysis of two other colostrum samples in the same way show similar results (data not shown), confirming good stability of the apelin peptides in bovine colostrum from sampling to analysis, and at least for 24-h storage at 4 °C. This result confirms that apelin peptides identified in this study are not artifactually produced during sample processing, and thus result from in vivo processes. Interindividual Variability of Bovine Colostrum Apelin Peptides

Variability was observed between the colostrums from 10 different cows analyzed regarding the nature and abundance of the apelin peptides detected (Figure 4). The occurrences of the apelin peptides in the 10 colostrums analyzed are summarized in Table 1. For instance, only 11 apelin peptides were detected in all the colostrums analyzed (i.e., apelin-52/F, -36, -36/F, -36/PF, -35, -35/F, -35/MPF, -31/F, -28/F, -28/PF, -17/F, Table 1), whereas apelin-37, -25, -25/F, -25/PF, -23 and -15/F were detected only in one colostrum (Table 1). Moreover, the proportions of apelin peptides fluctuated between colostrums. Although apelin-

28/F and 35/F appeared as the most intense apelin peptides in 50 and 30% of the colostrums tested, respectively, the relative intensities of these 2 peptides ranged from 0.3 in colostrums from cow 8 to 10.9 in colostrum from cow 10 (Figure 4). Thus, the presence and the proportion of the apelin peptides in the colostrums from 10 different cows seemed highly variable. A recent study has demonstrated the presence of both proteases and protease inhibitors in bovine colostrum. It could be hypothesized that individual imbalance in the expression or secretion of proteases and protease inhibitors detected in colostrum23 might lead to the variable apelin peptide patterns observed. Apelin Peptide Family: From Colostrum to Milk Samples

The protein compositions of colostrum and milk are known to be different, with proteins exclusively represented either in milk or in colostrum along with both up- and down-regulated species.23 The global concentration of apelin peptides in milk has been reported to decrease markedly during the first days after parturition.20 To evaluate changes in apelin peptide composition from colostrum to mature milk, samples from one cow were obtained on days 0 (colostrum), 5228



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Figure 5. Change in apelin peptide composition from bovine colostrum to milk. Synthetic stable isotope-labeled human apelin-36 was spiked at 50 ng/mL before extraction and used for normalization of all other signals (relative intensity). (A) Milk samples from cow 1 were collected on day 0 (colostrum), day 2 (early milk) and day 8 (mature milk) and analyzed by FTMS. (B) Mature milk samples from two individual cows and from a 300-cow livestock pool were collected and analyzed by FTMS. (C) Commercially available pasteurized milk and sterilized milk were purchased at a local supermarket and analyzed by FTMS.

2 (early milk) and 8 (mature milk) after parturition and analyzed as previously described (Figure 5A). A huge decrease of the C-terminal truncated forms along with a significant increase of the intact form was observed in early milk, and more markedly in mature milk, compared with colostrum. As an illustration, apelin-35/F relative intensities varied between day 0 and day 8 from 54 to 0% versus from 6 to 26% for apelin-55. These results suggest a change in apelin peptide composition during the first week of milking. In addition, 2 other individual mature milk samples, as well as livestock mature milk (pool of 300 individual mature milk samples) were analyzed (Figure 5B). The mature milk samples all contained prominent intact apelin-55 and -22, and to a lesser extent apelin36, -31, -28, -22, -18, -16, -15, and -14. Only minor C-terminal truncated forms could be observed, except apelin-17/F, which was the second most intense form in mature milk from cows 11, 12 and the livestock pool. Thus, apelin peptides in mature milk seem to be qualitatively constant. Altogether, these results suggest that apelin peptide composition in colostrum evolves quickly during the first week postpartum and stabilizes in mature milk. While the apelin peptide family mainly comprises N- and

C-terminal truncated forms in colostrum, mature milk contains preponderant intact apelin-55 and -22. The biological significance of these composition changes requires further study. Digestive functions in the calf are not fully effective at birth and reduced gastrointestinal enzymatic activities during the first 48 h of extrauterine life have been reported.30 As short apelin peptides have more potent effects on the APJ receptor,4 proapelin processing in colostrum upon arrival in the newborn stomach might promote their action in the developing gastrointestinal tract. Two days and, more markedly, seven days after parturition, digestive function in the calf is better developed.30 At this developing stage, it could be hypothesized that proapelin provided by milk is effectively processed in the calf gastrointestinal tract. Occurrence of Apelin Peptides in Commercial Milk Samples

The presence of apelin peptides has been reported in commercial bovine milk samples,20 but the nature of these peptides has not been studied. The pattern of apelin peptides in pasteurized milk appears to be similar to that of mature milk (Figure 5C). Thus, apelin peptide composition in milk does not seem to be 5229


Journal of Proteome Research affected by pasteurization. However, ultrahigh temperature sterilization results in a 15-fold increase in apelin-17/F relative intensity, compared with pasteurized milk (Figure 5C). Sterilization is done at twice the temperature of pasteurization (∼140 °C versus ∼70 °C) and is known to modify protein characteristics31 and may promote the formation of the apelin-17/F. The implications for human health of the presence of apelin peptides in commercially available milk are currently unknown and deserve further attention.

’ CONCLUDING REMARKS Bovine colostrum contains a wide range of apelin peptides at high concentrations, but none have yet been rigorously characterized.3,20 We have successfully implemented an untargeted LC MS method combining FTMS measurements and sequential MSn experiments to identify and monitor 46 apelin forms in bovine colostrums and milk. Our findings show that there is in vivo release of multiple and variable apelin peptides in bovine colostrum, and that apelin peptide composition changes during milk maturation to a more constant and uniform composition in mature milk. There is a need to investigate the physiological impact of these changes on the developing calf. The effect on human health of the apelin peptides ingested with commercial bovine milk should also be addressed. In this way, using the method presented here we have analyzed human colostrum and milk samples, but no apelin peptide was detected (data not shown). Global apelin peptide concentrations previously reported in this matrix using bio-20 or immunoassays21 ranged from 0.5 to 5 ng/mL. Considering that the estimated sensitivity of our method is 0.5 ng/mL, a wide diversity of apelin peptides in human colostrum and milk could mean that individual apelin peptides are present at low concentrations, thereby explaining the absence of detectable signals. Development of more sensitive methods is currently ongoing to investigate apelin peptides in human colostrum and milk.

’ AUTHOR INFORMATION Corresponding Author

*CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, 911191 Gif-sur-Yvette. Tel: 00-33-(0)4-66-79-19-04. Fax: 0033-(0)4-66-79-19-08. E-mail: [email protected].

’ ACKNOWLEDGMENT We thank Sophie Cholet and Jean-Michel Hennequin for their help. ’ REFERENCES (1) Tatemoto, K.; Hosoya, M.; Habata, Y.; Fujii, R.; et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun. 1998, 251, 471–476. (2) Lee, D. K.; Cheng, R.; Nguyen, T.; Fan, T.; et al. Characterization of apelin, the ligand for the APJ receptor. J. Neurochem. 2000, 74, 34–41. (3) Hosoya, M.; Kawamata, Y.; Fukusumi, S.; Fujii, R.; et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J. Biol. Chem. 2000, 275, 21061–21067. (4) Kleinz, M. J.; Davenport, A. P. Emerging roles of apelin in biology and medicine. Pharmacol. Ther. 2005, 107, 198–211. (5) Foldes, G.; Horkay, F.; Szokodi, I.; Vuolteenaho, O.; et al. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure. Biochem. Biophys. Res. Commun. 2003, 308, 480–485.

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Identification and Characterization of Apelin Peptides in Bovine

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