Quantitative proteomic analysis of eight cartilaginous tissues reveals characteristic differences as well as similarities between subgroups

Abstract

Human synovial joints display a characteristic anatomic distribution of arthritis, e.g. rheumatoid arthritis primarily affects the metacarpophalangeal and proximal finger joints, but rarely the distal finger joints, whereas osteoarthritis occurs in the distal and proximal finger joints. Pelvospondylitis has a selective localization to the spine and sacroiliac joints. Is this tropism due to differences between the cartilages at the molecular level? To substantiate this concept the present study provides a background detailed compositional analysis by relative quantification of extracellular matrix proteins in articular cartilages, meniscus, intervertebral disc, rib, and tracheal cartilages on samples from 5-6 different individuals using an optimized approach for proteomics. Tissue extraction followed by trypsin digestion and two-dimensional LC separations coupled to tandem mass spectrometry, relative quantification with isobaric labeling, iTRAQ(TM), was used to compare the relative abundance of about 150 proteins. There were clear differences in protein patterns between different kinds of cartilages. Matrilin-1 and epiphycan were specific for rib and trachea, whereas asporin was particularly abundant in the meniscus. Interestingly, lubricin was prominent in the intervertebral disc, especially in the nucleus pulposus. Fibromodulin and lumican showed distributions that were mirror images of one other. Analyses of the insoluble residues from guanidine extraction revealed that a fraction of several proteins remained unextracted, e.g. asporin, CILP, and COMP, indicating cross-linking. Distinct differences in protein patterns may relate to different tissue mechanical properties, and to the intriguing tropism in different patterns of joint pathology.

FIGURE 1.

MS-MS spectrum of the peptide ion m/z = 484.74Da (2+) illustrating the multiplex method. Samples from the meniscus, M (label 114), annulus fibrosus, AF (115), and nucleus pulposus, NP (116) were mixed with a reference sample (117) and the individual low mass reporter signals (114–117) are shown in the inset for the relative quantification. The other fragment ions are used for the identification using a database search in this case identifying the asporin peptide AFLTTK.

FIGURE 2.

Extractability of selected proteins. Extractability is defined as the percentage recovered in extracts using 4 m GdnHCl compared with the sum of amounts in extract + residue, the latter released upon trypsin digestion of the extraction residue, error bars indicate the S.D. at p = 0.05. Selected tissues include femoral head, humeral head, tibial knee articular cartilages, meniscus, annulus fibrosus, nucleus pulposus, rib, and tracheal cartilages.

FIGURE 3.

Average ratios of the individual proteins aggrecan, lubricin and matrilin-1 in various tissues. Tissues include articular cartilage from femoral head, humeral head, and tibial knee condyle as well as samples from meniscus, annulus fibrosus, nucleus pulposus, rib, and tracheal cartilages. Error bars are calculated using the confidence interval at p = 0.05 and n = 5 (n = 6 for rib and trachea).

FIGURE 4.

Relative protein abundance pattern, in cartilaginous tissues, of SLRPs family. The SLRPs of class I are shown in a, class II in b, and class III and IV in c. Error bars are calculated using the confidence interval at p = 0.05 and n = 5 (n = 6 for rib and tracheal cartilages). The Western blot insets for asporin and chondroadherin show bands with intensities that correlate with the iTRAQ data.

FIGURE 5.

Proteins significantly different (p = 0.01) in abundance between femoral head (F) and tibial knee (K) articular cartilage using paired t tests. High protein abundances are indicated in yellow, whereas low abundances are in blue. Samples with the same number, e.g. F7 and K7, correspond to the same individual.