International Orthopaedics

, Volume 31, Issue 6, pp 743–751

Detection of bone and cartilage-related proteins in plasma of patients with a bone fracture using liquid chromatography–mass spectrometry

Authors

  • Lovorka Grgurevic
    • Laboratory of Mineralized Tissues, School of MedicineUniversity of Zagreb
  • Boris Macek
    • Department of Proteomics and Signal TransductionMax-Planck-Institute for Biochemistry
  • Dragan Durdevic
    • Clinic of Traumatology
    • Laboratory of Mineralized Tissues, School of MedicineUniversity of Zagreb
Original Paper

DOI: 10.1007/s00264-007-0404-z

Cite this article as:
Grgurevic, L., Macek, B., Durdevic, D. et al. International Orthopaedics (SICO (2007) 31: 743. doi:10.1007/s00264-007-0404-z
  • 145 Views

Abstract

Following bone fracture, a large number of growth factors, cytokines, and their cognate receptors involved in the repair process are active at the fracture site. To determine whether they appear in patients’ blood as candidate biomarkers for following the outcome of healing, we analysed the plasma of 25 patients with an acute bone fracture following affinity plasma purification, SDS gel electrophoresis and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Two hundred and thirteen nonredundant proteins were identified in the in-gel analysis of pooled plasma proteins. Gene ontology (GO) analysis indicated that a majority of detected proteins were of extracellular origin, whereas only a small number were of intracellular (cytosol and nucleus) origin. A significant proportion of detected proteins was involved in the cell growth and proliferation, transport and coagulation. Twelve proteins were potentially related to bone and cartilage metabolism, and several have not been previously identified in the plasma, including: TGF-β induced protein IG-H3, cartilage acidic protein 1, procollagen C proteinase enhancer protein and TGF-β receptor III.

Résumé

Après une fracture, un grand nombre de facteurs de croissance, cytokines et leurs récepteurs apparentés interviennent dans le processus de réparation des foyers de fracture. Nous avons analysé ces différents facteurs circulants chez 25 patients ayant présenté une fracture après purification du sang, électrophorèses, chromatographie et spectrographie de masse. 213 protéines ont été identifiées. L’analyse génétique de la majorité de ces protéines montre qu’elles sont d’origine extra cellulaires avec un très petit nombre de protéines intra cellulaires provenant notamment du noyau. Une proportion significative des protéines détectées intervient au niveau de la croissance, de la prolifération cellulaire et des phénomènes de coagulation. 12 protéines sont spécifiquement en rapport avec les métabolismes osseux et cartilagineux, plusieurs d’entre-elles n’avaient pas été préalablement identifiées au niveau du plasma comme la TGF-β, la protéine IG-H3, la CAP 1, le procollagène de type C, le TGF-β récepteur III.

Introduction

Blood is rich with a large amount of previously unstudied molecules that could reflect the ongoing physiological state of various tissues. As blood flows through most of the tissues of the human body the origins of plasma proteins are diverse. In the complex mixture of a plasma proteome, albumin and other carrier proteins, as well as proteins that originate from circulating blood cells, are present in a high abundance. Almost all cells in the body communicate directly or indirectly with blood and upon damage or cell death tissue-specific proteins are released into the bloodstream. Therefore, most potential undiscovered biomarkers will be eventually found in the plasma fraction, where much less abundant proteins enter the blood from the surrounding tissue.

Bone undergoes continuous turnover and remodelling consisting of bone formation and bone resorption, two opposite and well-balanced processes. The various bone serum and urinary markers are usually classified according to the metabolic process indicating low and high, decreased or increased bone turnover [1].

Following fracture, a large number of growth factors, cytokines, and their cognate receptors involved in bone repair are highly expressed at the fracture site in the first hours following injury. It is presumed that some or all of these factors initiate active repair process acting on the cells of the bone marrow, periosteum, and external soft tissues adjacent to the fracture site. Skeletal tissues are the main source of such proteins, while some are released from associated inflammatory cells at the site of injury [2, 10].

In this study we analysed proteins as candidate biomarkers expressed in the plasma of patients with an acute bone fracture. The plasma proteins of patients were characterised by SDS gel electrophoresis and affinity purification followed by tandem mass spectrometry LC-MS/MS. Following identification of proteins those associated with bone and cartilage metabolism were singled out. Some of characterised proteins have not yet been identified in the circulation and their presence or quantity could reflect the extent of injury and the success of the fracture repair.

Materials and methods

Plasma collection

Human blood plasma samples were supplied by the Clinic of Traumatology in Zagreb. The approval for the collecting samples was obtained from the institutional Ethics Committee. Blood samples from 25 adult humans (21–60 years of age) of both genders with a single long bone fracture were drawn into syringes containing 3.8% sodium citrate to form an anticoagulant-to-blood ratio (v/v) 1:9. Plasma was obtained by centrifugation (15 min at 3000xg), and aliquots of each adult blood sample were pooled for further analysis. Aliquot samples were stored at −80°C until analysis.

Affinity column purification

Pooled plasma of patients with a single-bone fracture (80 ml) was diluted twofold with 10 mM sodium phosphate buffer (pH 7) and applied to a heparin Sepharose column (Amersham Pharmacia Biotech), previously equilibrated with 10 mM sodium phosphate buffer (pH 7). Bound proteins were eluted from the column with 10 mM sodium phosphate buffer (pH 7) containing 1 M and 2 M NaCl. Eluted fractions were precipitated with saturated ammonium sulphate (SAS) to a final concentration of 35%.

SDS gel electrophoresis and in-gel digestion

SDS-PAGE was run on a NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, USA) using MOPS SDS buffer system, and subsequently stained with a Co-omassie staining kit (NuPAGE, Invitrogen), as instructed by the manufacturer. After staining, each of the seven gel lanes was sliced into 12 pieces and the corresponding pieces were combined as indicated in Fig. 1. The pieces were then subjected to in-gel reduction, alkylation and trypsin digestion as described previously [4]. Gel pieces were washed two times with acetonitrile/25 mM NH4HCO3, reduced by incubation with 10 mM dithiothreitol (DTT) for 45 minutes at 56°C and carboxyamidomethylated by incubation in 55 mM iodoacetamide for 45 minutes at room temperature. Trypsin (Promega) was added to dried gel pieces (150 ng per piece, diluted in 25 mM NH4HCO3) and incubated overnight at 37°C. Tryptic peptides were extracted with formic acid/acetonitrile/H2O (10:20:70); and 100% acetonitrile, dried and resuspended in trifluoroacetic acid/acetonitrile/H20 (1:2:97) for MS analysis.
https://static-content.springer.com/image/art%3A10.1007%2Fs00264-007-0404-z/MediaObjects/264_2007_404_Fig1_HTML.gif
Fig. 1

Pooled plasma protein separation by one-dimensional SDS gel. Pooled plasma of patients with a single-bone fracture was applied to a heparin Sepharose column (Amersham Pharmacia Biotech). Bound proteins were eluted from the column with 10 mM sodium phosphate buffer (pH 7) containing 1 M NaCl ( lane 4–7) and 2 M NaCl (lane 1–3), lane 8 molecular mass marker. The numbers in the column indicate gel lanes sliced and prepared for MS analysis. Gel was stained with a Comassie brilliant blue

Liquid chromatography–mass spectrometry

Tryptic peptides were analysed by liquid chromatography-mass spectrometry (LC-MS). Agilent 1100 nanoflow HPLC system (Agilent Technologies) was coupled to a LTQ-Orbitrap mass spectrometer (Thermo Fisher) using a nano-electrospray LC-MS interface (Proxeon Biosystems). Peptides were loaded on a home-made 75 μm C18 HPLC column in solvent “A” (0.5% acetic acid in Milli-Q water) and eluted with a 70-minute segmented linear gradient of 10–60% solvent “B” (80% acetonitrile, 0.5% acetic acid in Milli-Q water) at a flow rate of ca. 250 nL/min.

Mass spectrometer was operated in the positive ion mode. Each measurement cycle consisted of a full MS scan acquired in the orbitrap analyser at a resolution of 60,000, and MS/MS fragmentation of the five most intense ions in the linear ion trap analyser. To further improve mass accuracy, the lock-mass option was used as described previously [9]. This has resulted in a typical peptide average absolute mass accuracy of less than 1 ppm.

Peak lists were generated using in-house developed software (Raw2msm) [9], and searched against concatenated forward and reverse (“decoy”) IPI human database (version 3.13) using Mascot search engine (Matrix Science). Searches were done with trypsin specificity (two missed cleavages allowed), carboxyamidomethylation as fixed modification, and oxidised methionine as variable modification. Precursor ion and fragment ion mass tolerances were 10 ppm and 0.5 Da, respectively.

Results of the database search were validated in the MSQuant software (http://msquant.sourceforge.net). Only peptides with a mass deviation lower than 5 ppm were accepted; two peptides were required for protein identification.

Gene ontology (GO) analysis was performed using ProteinCenter software package (Proxeon Biosystems).

Results

Gene ontology analysis of characterised plasma proteins

Pooled plasma samples were subjected to heparin affinity chromatography to enrich for proteins specific for bone and cartilage, majority of which are known to have heparin binding domains. This has also partially removed highly abundant plasma proteins, such as albumin, immunoglobulins, transferin and haptoglobulin. Fractions of interest were collected, precipitated with ammonium sulphate and separated on 1D SDS-PAGE gel (Fig. 1). Gel bands were excised, digested with trypsin and analysed by LC-MS/MS. Peptide fragmentation spectra were searched against the human IPI protein database, and the results of the database search were validated using MSQuant software. Only peptides with a mass deviation lower than 5 ppm were accepted; two peptides were required for protein identification, which led to an overall false-positive rate of less than 1% at both the peptide and the protein level.

In total, 213 nonredundant proteins were identified in the in-gel analysis of pooled plasma proteins from patients with a single bone fracture and listed in Table 1.
Table 1

Proteins identified by a tandem mass spectrometry LC-MS/MS in pooled purified plasma from patients with fracture

IPI Accession number

Protein name

--IPI00000137.1

N-acetylglucosamine-1-phosphotransferase subunit gamma precursor

IPI00000138.1

Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

IPI00000816.1

14-3-3 protein epsilon

IPI00001753.1

Myosin-4

IPI00002147.4

Chitinase-3-like protein 1 precursor

IPI00003176.1

Serine protease HTRA1 precursor

IPI00003351.2

Extracellular matrix protein 1 precursor

IPI00003590.2

Isoform 1 of Sulfhydryl oxidase 1 precursor

IPI00004957.1

Angiopoietin-related protein 3 precursor

IPI00006114.4

Pigment epithelium-derived factor precursor

IPI00006154.1

Isoform Long of Complement factor H-related protein 2 precursor

IPI00006510.1

Tubulin beta-1 chain

IPI00006543.2

Complement factor H-related 5

IPI00006662.1

Apolipoprotein D precursor

IPI00007118.1

Plasminogen activator inhibitor 1 precursor

IPI00007199.4

Protein Z-dependent protease inhibitor precursor

IPI00007221.1

Plasma serine protease inhibitor precursor

IPI00007858.1

Myosin-13

IPI00008556.1

Isoform 1 of Coagulation factor XI precursor

IPI00009865.1

Keratin, type I cytoskeletal 10

IPI00009920.2

Complement component C6 precursor

IPI00010295.1

Carboxypeptidase N catalytic chain precursor

IPI00010779.4

Tropomyosin 4

IPI00010896.2

IPI00011252.1

Complement component C8 alpha chain precursor

IPI00011261.2

Complement component C8 gamma chain precursor

IPI00011264.1

Complement factor H-related protein 1 precursor

IPI00013004.1

Isoform 1 of Pyridoxal kinase

IPI00016915.1

Insulin-like growth factor-binding protein 7 precursor

IPI00017256.5

Ras suppressor protein 1

IPI00017530.1

Ficolin-2 precursor

IPI00017696.1

Complement C1s subcomponent precursor

IPI00018219.1

Transforming growth factor-beta-induced protein ig-h3 precursor

IPI00018305.3

Insulin-like growth factor-binding protein 3 precursor

IPI00019359.3

Keratin, type I cytoskeletal 9

IPI00019579.1

Complement factor D precursor

IPI00019580.1

Plasminogen precursor

IPI00019581.1

Coagulation factor XII precursor

IPI00019591.1

Isoform 1

IPI00020091.1

Alpha-1-acid glycoprotein 2 precursor

IPI00020996.3

Insulin-like growth factor-binding protein complex acid labile chain precursor

IPI00021263.3

IPI00021304.1

Keratin, type II cytoskeletal 2 epidermal

IPI00021364.1

Properdin precursor

IPI00021439.1

Actin, cytoplasmic 1

IPI00021440.1

IPI00021727.1

C4b-binding protein alpha chain precursor

IPI00021841.1

IPI00021842.1

Apolipoprotein E precursor

IPI00021854.1

Apolipoprotein A-II precursor

IPI00021885.1

Isoform 1 of Fibrinogen alpha chain precursor

IPI00021891.5

Isoform Gamma-B of Fibrinogen gamma chain precursor

IPI00022200.2

alpha 3 type VI collagen isoform 1 precursor

IPI00022229.1

Apolipoprotein B-100 precursor

IPI00022371.1

Histidine-rich glycoprotein precursor

IPI00022391.1

Serum amyloid P-component precursor

IPI00022392.1

Complement C1q subcomponent subunit A precursor

IPI00022394.2

Com

IPI00022395.1

Complement component C9 precursor

IPI00022418.1

Isoform 1 of Fibronectin precursor

IPI00022426.1

AMBP protein precursor

IPI00022434.2

ALB protein

IPI00022488.1

Hemopexin precursor

IPI00022822.4

Isoform Long of Collagen alpha-1(XVIII) chain precursor

IPI00022895.7

Alpha-1B-glycoprotein precursor

IPI00022937.3

Coagulation factor V

IPI00023006.1

Actin, alpha cardiac muscle 1

IPI00023728.1

Gamma-glutamyl hydrolase precursor

IPI00024825.2

Isoform A of Proteoglycan-4 precursor

IPI00025204.1

CD5 antigen-like precursor

IPI00025276.1

Isoform XB of Tenascin-X precursor

IPI00025862.1

C4b-binding

IPI00026314.1

Isoform 1 of Gelsolin precursor

IPI00026689.4

Hypothetical protein DKFZp686L20222

IPI00027507.1

Complement factor H-related protein 3 precursor

IPI00027780.1

72 kDa type IV collagenase precursor

IPI00027827.2

Extracellular superoxide dismutase [Cu-Zn] precursor

IPI00028091.2

Actin-like protein 3

IPI00028413.7

Inter-alpha-trypsin inhibitor heavy chain H3 precursor

IPI00029061.2

Selenoprotein P precursor

IPI00029193.1

Hepatocyte growth factor activator precursor

IPI00029236.1

Insulin-like growth factor-binding protein 5 precursor

IPI00029739.4

Isoform 1 of Complement factor H precursor

IPI00029863.4

Alpha-2-antiplasmin precursor

IPI00032179.2

Antithrombin III variant

IPI00032220.3

Angiotensinogen precursor

IPI00032258.4

IPI00032291.1

Complement C5 precursor

IPI00032292.1

Metalloproteinase inhibitor 1 precursor

IPI00032311.4

Lipopolysaccharide-binding protein precursor

IPI00032328.1

Isoform HMW of Kininogen-1 precursor

IPI00041065.3

Hyaluronan-binding protein 2 precursor

IPI00043083.1

Beta-parvin

IPI00060715.1

BTB/POZ domain-containing protein KCTD12

IPI00154742.5

25 kDa protein

IPI00163207.1

Isoform 1 of N-acetylmuramoyl-L-alanine amidase precursor

IPI00164623.4

187 kDa protein

IPI00165438.2

Muscle type neuropilin 1

IPI00168728.1

FLJ00385 protein (Fragment)

IPI00178083.2

29 kDa protein

IPI00183968.4

tropomyosin 3 isoform 1

IPI00186903.3

Isoform 2 of Apolipoprotein-L1 precursor

IPI00216134.3

tropomyosin 1 alpha chain isoform 7

IPI00216699.1

Isoform 2 of Unc-112-related protein 2

IPI00216773.4

ALB protein

IPI00218192.2

Isoform 2 of Inter-alpha-trypsin inhibitor heavy chain H4 precursor

IPI00218732.2

Serum paraoxonase/arylesterase 1

IPI00219018.6

Glycer

IPI00219465.4

Transcobalamin-2 precursor

IPI00219682.5

Erythrocyte band 7 integral membrane protein

IPI00219713.1

Isoform Gamma-A of Fibrinogen gamma chain precursor

IPI00220327.2

Keratin, type II cytoskeletal 1

IPI00220350.1

Isoform Beta-3B of Integrin beta-3 precursor

IPI00220642.6

14–3–3 protein gamma

IPI00220644.8

pyruvate kinase 3 isoform 2

IPI00220701.3

Isoform 2 of Collagen alpha-3(VI) chain precursor

IPI00289831.4

Isoform PTPS of Receptor-type tyrosine-protein phosphatase S precursor

IPI00291262.3

Clusterin precursor

IPI00291866.5

Plasma protease C1 inhibitor precursor

IPI00291867.3

Complement factor I precursor

IPI00292218.3

Hepatocyte growth factor-like protein precursor

IPI00292530.1

Inter-alpha-trypsin inhibitor heavy chain H1 precursor

IPI00292950.4

Heparin cofactor 2 precursor

IPI00293925.2

Isoform 1 of Ficolin-3 precursor

IPI00294004.1

Vitamin K-dependent protein S precursor

IPI00294193.4

Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H4 precursor

IPI00294395.1

Complement component C8 beta chain precursor

IPI00295976.5

Isoform 1 of Integrin alpha-IIb precursor

IPI00296099.6

Thrombospondin-1 precursor

IPI00296165.5

Complement C1r subcomponent precursor

IPI00296176.2

-

IPI00296537.3

Isoform C of Fibulin-1 precursor

IPI00296608.6

Complement component C7 precursor

IPI00297284.1

Insulin-like growth factor-binding protein 2 precursor

IPI00297550.7

Coagulation factor XIII A chain precursor

IPI00297779.6

T-complex protein 1 subunit beta

IPI00298497.3

Fibrinogen beta chain precursor

IPI00298828.3

Beta-2-glycoprotein 1 precursor

IPI00298860.5

Growth-inhibiting protein 12

IPI00298971.1

Vitronectin precursor

IPI00298994.5

271 kDa protein

IPI00299145.8

Keratin, type II cytoskeletal 6E

IPI00299547.4

Neutrophil gelatinase-associate

IPI00299738.1

Procollagen C-endopeptidase enhancer 1 precursor

IPI00302592.2

filamin 1

IPI00303476.1

ATP synthase subunit beta, mitochondrial precursor

IPI00303963.1

C

IPI00304273.2

Apolipoprotein A-IV precursor

IPI00304865.3

transforming growth factor, beta receptor III

IPI00305461.2

Inter-alpha-trypsin inhibitor heavy chain H2 precursor

IPI00306311.8

Pleckstrin

IPI00328609.3

Kallistatin precursor

IPI00328703.1

OAF homolog

IPI00329775.7

Isoform 1 of Carboxypeptidase B2 precursor

IPI00333828.4

Serpin A11 precursor

IPI00339228.1

Isoform 8 of Fibronectin precursor

IPI00382436.1

Ig lambda chain V-III region SH

IPI00382606.1

Factor VII active site mutant immunoconjugate

IPI00383111.2

57 kDa protein

IPI00384280.5

Prenylcysteine oxidase precursor

IPI00384444.4

Keratin, type I cytoskeletal 14

IPI00384938.1

Hypothetical protein DKFZp686N02209

IPI00385429.1

collectin sub-family member 11 isoform b

IPI00387025.1

Ig kappa chain V-I region DEE

IPI00387099.1

Ig kappa chain V-I region Rei

IPI00387113.1

Ig kappa chain V-III region B6

IPI00387120.1

Ig kappa chain V-IV region Len

IPI00399007.5

Hypothetical protein DKFZp686I04196 (Fragment)

IPI00418153.1

Hypothetical protein DKFZp686I15212

IPI00418163.3

complement com

IPI00418495.4

Platelet

IPI00419424.3

IGKV1-5 protein

IPI00426051.3

Hypothetical protein DKFZp686C15213

IPI00430808.1

Hypothetical protein

IPI00430842.3

IGHA1 protein

IPI00431645.1

HP protein

IPI00448925.3

IGHG1 protein

IPI00451624.1

Isoform 1 of Cartilage acidic protein 1 precursor

IPI00465248.5

enolase 1

IPI00465378.1

Apolipoprotein A-V precursor

IPI00465439.4

-

IPI00472073.1

HLA class I histocompatibility antigen, B-59 alpha chain precursor

IPI00472610.2

IGHM protein

IPI00477090.5

IGHM protein

IPI00477597.1

Isoform 1 of Haptoglobin-related protein precursor

IPI00477644.2

26 kDa protein

IPI00477992.1

complement component 1, q subcomponent, B chain precursor

IPI00478003.1

Alpha-2-macroglobulin precursor

IPI00478493.3

HP protein

IPI00479116.1

Carboxypeptidase N subunit 2 precursor

IPI00479708.5

IGHM protein

IPI00549291.4

IGHM protein

IPI00550991.3

Isoform 1 of Alpha-1-antichymotrypsin precursor

IPI00553177.1

Alpha-1-antitrypsin

IPI00641368.4

Tsukushi precursor

IPI00641737.1

Haptoglobin precursor

IPI00643034.2

Isoform 1 of Phospholipid transfer protein precursor

IPI00643041.2

GTP-binding nuclear protein Ran

IPI00643525.1

Complement component 4A

IPI00646909.2

Tubulin alpha-8 chain

IPI00654888.3

Kallikrein B, plasma (Fletcher factor) 1

IPI00719373.1

IGLC1 protein

IPI00745872.2

Isoform 1 of Serum albumin precursor

IPI00783024.1

Myosin-reactive immunoglobulin heavy chain variable region (Fragment)

IPI00783987.1

Complement C3 precursor (Fragment)

IPI00784822.1

Hypothetical protein

IPI00785050.1

Hypothetical protein

IPI00785200.1

Hypothetical protein

IPI00787629.1

similar to Apolipoprotein

IPI00790993.1

104 kDa protein

IPI00794487.1

Immunoblobulin light chain (Fragment)

IPI00807531.1

IGHG1 protein

Gene ontology (GO) analysis of plasma proteins showed that a majority (63.8%) of detected proteins were of extracellular origin, whereas only a small number (7.5%) were of intracellular (cytosol and nucleus) origin. Interestingly, we also detected a relatively high number (35.2%) of membrane related proteins (Fig. 2a).
https://static-content.springer.com/image/art%3A10.1007%2Fs00264-007-0404-z/MediaObjects/264_2007_404_Fig2_HTML.gif
Fig. 2

Protein categorisation using gene ontology (GO) component terms. Total nonredundant proteins identified from pooled plasma of patients with a single bone fracture were compared according to the following categories: (A) subcellular localisation, (B) molecular function and (C) biological process

According to the molecular function analysis, 37.6% of detected proteins had catalytic properties, 18.3% were classified as signal transducers, and 13.1% as transporters (Fig. 2b).

In terms of biological activity, a significant proportion of detected proteins were involved in the cell growth and proliferation (21.1%), transport (23.9%) and coagulation (13.1%) (Fig. 2c).

Identification of bone- and cartilage-related proteins

From the list of detected proteins we singled out 12 proteins which could be related to bone and cartilage metabolism (Table 2). Among them there were proteins not previously identified in the plasma, like cartilage acidic protein 1 (CRTAC-1), which were identified with 28 peptides and an average peptide Mascot score of 53. A molecule also related to the cartilage metabolism was the Splice isoform A of the proteoglycan-4 or lubricin, identified with two peptides and an average peptide Mascot score of 60.
Table 2

The 12 proteins involved in bone and cartilage metabolism, their peptides, and Mascot score

Protein name

IPI accession number

Number of identified peptides

Average peptides Mascot score

Previously identified in plasma

GO console: molecular function

GO console: cellular component

GO console: biological process

Extracellular matrix protein 1 precursor

IPI00003351.2

57

54

+

Signal transducer activity

Extracellular

Cell communication

Structural molecule activity

Metabolism

Transporter activity

Regulation of biological process

Transport

Transforming growth factor beta induced protein IG-H3 precursor

IPI 00018219.1

20

57

Protein binding

Extracellular

Cell proliferation

  

Regulation of biological process

  

Sensory perception

Splice isoform 1 of cartilage acidic protein 1 precursor

IPI00451624.1

28

53

Metal ion binding

Proteasom

 

Golgi complex

Splice isoform 2 of collagen alpha 3 (VI) chain precursor

IPI00220701.3

10

62

+

Enzyme regulator activity

Extracellular

Development

Protein binding

Transport

Structural molecule activity

 

Type IV collagenase precursor

IPI00027780.1

3

74

+

Catalytic activity

Extracellular

Development

Enzyme regulator activity

Metabolism

Metal ion binding

Alpha 3 type VI collagen isoform 1 precursor

IPI00022200.2

2

60

Enzyme regulator activity

Extracellular

Development

Protein binding

Transport

Structural molecule activity

Procollagen C proteinase enhancer protein precursor

IPI00299738.1

10

58

Nucleic acid binding

Extracellular

Development

Protein binding

Metabolism

Transforming growth factor beta receptor III

IPI00304865.3

4

44

Receptor activity

Golgi

Cell communication

Signal transducer activity

Development

Isoform Long of Collagen alpha-1(XVIII) chain precursor

IPI00022822.4

5

36

+

Metal ion binding

Extracellular

Cell death

Protein binding

Cell motility

Structural molecular activity

Cell organization and biogenesis

Cell proliferation

Development

Regulation of biological process

Sensory perception

Transport

Hyaluronan binding protein 2 precursor

IPI00041065.3

7

51

+

Catalytic activity

Extracellular

Metabolism

Metalloproteinase inhibitor 1 precursor

IPI00032292.1

5

49

+

Catalytic activity

Extracellular

Cell proliferation

Enzyme regulator activity

Development

Metal ion binding

Regulation of biological process

Metabolism

Splice isoform A of proteoglycan-4 precursor

IPI00024825.2

2

60

+

 

Extracellular

Cell proliferation

Individual peptide Mascot scores >27 indicate identity or extensive homology (p < 0.05).

Transforming growth factor beta receptor III was identified in the plasma for the first time with four specific peptides and an average peptide Mascot score of 44, as well as the transforming growth factor beta-induced protein IG-H3, with 20 peptides and an average peptide Mascot score of 57.

Among extracellular matrix proteins not previously detected in the plasma was the alpha 3 type VI collagen isoform 1 identified with two peptides and an average peptide Mascot score of 60.

Previously identified extracellular matrix proteins of interest for bone repair included: isoform long of collagen alpha-1 (XVIII) chain precursor or endostatin with 5 peptides and an average peptide Mascot score of 36, splice isoform 2 of collagen alpha 3 (VI) chain precursor with 10 identified peptides and an average peptide Mascot score of 62, extracellular matrix protein 1 precursor with 57 identified peptides and an average peptide Mascot score of 54, and type IV collagenase precursor or matrix metalloproteinase-2 (MMP2) with 3 identified peptides and an average peptide Mascot score of 74 (Table 2). MMP-2 degrades extra-cellular proteins and disrupts the subendothelial basement membrane, thus enabling the transmigration of inflammatory cells. Another metalloproteinase inhibitor 1 (TIMP-1) was identified with five peptides and an average peptide Mascot score of 49 (Table 2).

Discussion

In this study we used state-of-the art proteomics approach, based on high accuracy mass spectrometry, to characterise proteins in the plasma of patients with an acute bone fracture. Gene ontology showed a variety of different proteins, among which several have not been previously detected in the blood and could reflect the bone and cartilage stages of bone regeneration. Among them CRTAC-1, a glycosylated extracellular matrix molecule secreted by chondrocytes from the human articular cartilage. In the cell culture it was described as a candidate marker to distinguish the chondrocyte-like phenotype and activity from osteoblast-like and mesenchymal stem cells [15]. Thus its presence in the plasma of patients with an acute fracture could indicate the normal development and function of cartilaginous callus formation within the first week after the fracture and then its replacement by bone in the following weeks. In parallel CRTAC-1 could also indicate a concomitant joint cartilage injury immediately following an accident. In this way it may help distinguish between fractures with and without damaged joint cartilage, which would make CRTAC-1 an ideal marker for the various stages of the fracture repair. In the following study we need a precise time-related follow up of the plasma profile of CRTAC-1 in patients with a bone fracture with and without injured joint cartilage.

Splice isoform A of the proteoglycan-4 is a secreted, cytoprotective glycoprotein, a product of the gene proteoglycan 4 and a major component of the synovial fluid participating in the boundary lubrication of synovial fluids [1113]. It prevents protein deposition onto cartilage from synovial fluid, controls the adhesion-dependent synovial growth, and inhibits the adhesion of synovial cells to the cartilage surface [7]. It is highly expressed by synoviocytes and could serve as a marker of their activity following injury. It has been previously identified in the plasma [5].

The fracture healing process might be associated with a distinctive enzyme activity pattern at the fracture site, which may be reflected in their respective plasma/serum concentrations of various enzymes in their activity pattern. Thus, variations in the concentration of TIMP-1 and MMP-1 in the period following the fracture could have an important influence on the bone healing, as well as on other mechanisms leading to the development of a nonunion [6].

Discovery of circulating TβRIII was surprising since it is known that it has an essential role in the murine and chick development and that TβRIII knockout mice have an embryonic lethal phenotype. TβRIII acts as a TGF-β co-receptor, concentrating ligand on the cell surface and enhancing ligand binding to the signalling TGF-β receptor TβRII [8]. It is well known that transforming growth factor β1 (TGF-β1) and its receptor TβRII together with extracellular matrix proteins osteocalcin and collagen type I have an important role in the process of fracture healing. This result might add TβRIII to a list of novel biomarkers for following fracture repair. Recently, it was shown that TβRIII has also an important function as a suppressor of breast and prostate cancer progression [3, 16]. The possibility of following the cancer progression by detection of TßRIII in plasma should be further examined, especially knowing the role of TGFβ-1 and related family members in the progression of tumour growth and metastasis [14].

TGF-β IG-H3 adhesion protein in plasma may play an important role in the cell-collagen interactions and binding to type I, II and IV collagens and may have an important role in the endochondral bone formation. It may also serve as a potential biomarker for the progression of successful bone healing. Additional studies will be needed to demonstrate the potential of these newly discovered plasma proteins as potential biomarkers for following the fracture healing and related disorders in humans.

Copyright information

© Springer-Verlag 2007