Collagen Sequence Research Articles

Mapping Extracellular Matrix Proteins in Formalin-Fixed, Paraffin-Embedded Tissues by MALDI Imaging Mass Spectrometry.

Collagens and elastin form the fundamental framework of all tissues and organs, and their expression and post-translational processing are tightly regulated in disease and health. Because of their unique structural composition and properties, it is a recognized challenge to access these protein structures within the complex tissue microenvironment to understand how localized changes modulate tissue health. We describe a new workflow using a combination of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) with matrix metalloproteinase (MMP) enzymes to access and report on spatial localization of collagen and elastin sequences in formalin-fixed, paraffin-embedded (FFPE) tissues. The developed technology provides new access to collagens and elastin sequences localized to tissue features that were previously unattainable. This high-throughput technological advance should be applicable to any tissue regardless of disease type, tissue origin, or disease status and is thus relevant to all research: basic, translational, or clinical.

Engineered Collagen: A Redox Switchable Framework for Tunable Assembly and Fabrication of Biocompatible Surfaces.

Collagen, processed into several morphologies and originating from various sources, has long since been used as a biocompatible material that can assist wound healing and tissue regeneration. With the advent of biotechnology and solid-phase peptide synthesis, new possibilities arise to create rationally designed biomaterials based on collagen sequences incorporating new functionalities while maintaining the beneficial properties of natural collagen. In this study a new class of synthetic collagen materials is presented, defined by its simplistic core structure and its therefore predictable behavior. These so-called eCols (engineered collagens) consist of a varying number of Gly-Pro-Pro repeats, a redox-switchable aminoterminal nucleation site and an optional carboxyterminal cell adhesion motif. We show which of these proteins are able to self-assemble into triple helices and cross-linked gelatinous networks and provide insights into their cytocompatibility in vitro.

WS2 and MoS2 biosensing platforms using peptides as probe biomolecules

Biosensors based on the two-dimensional layered nanomaterials transition metal dichalcogenides such as WS2 and MoS2 have shown broad applications, while they largely rely on the utilization of single stranded DNA as probe biomolecules. Herein we have constructed novel WS2- and MoS2- based biosensing platforms using peptides as probe biomolecules. We have revealed for the first time that the WS2 and MoS2 nanosheets display a distinct adsorption for Arg amino acid and particularly, Arg-rich peptdies. We have demonstrated that the WS2 and MoS2 dramatically quench the fluorescence of our constructed Arg-rich probe peptide, while the hybridization of the probe peptide with its target collagen sequence leads to the fluorescence recovery. The WS2-based platform provides a sensitive fluorescence-enhanced assay that is highly specific to the target collagen peptide with little interferences from other proteins. This assay can be applied for quantitative detection of collagen biomarkers in complex biological fluids. The successful development of WS2- and MoS2- based biosensors using non-ssDNA probes opens great opportunities for the construction of novel multifunctional biosensing platforms, which may have great potential in a wide range of biomedical field.

Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide

The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG)8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions.

Sol–gel synthesis of collagen-inspired peptide hydrogel

Conceiving biomaterials able to mimic the specific environments of extracellular matrices are a prerequisite for tissue engineering applications. Numerous types of polymers (PEG, PLA, etc.) have been used for the design of biocompatible scaffolds, but they are still less efficient than natural biopolymers such as collagen extracts. Chemically modified and loaded with different bioactive factors, biopolymers afford an environment favourable to cell proliferation and differentiation. Unfortunately, they present several drawbacks, such as weak batch-to-batch reproducibility, potential immunogenicity and high cost of production. Herein we propose a fully synthetic covalent hydrogel obtained by sol–gel polymerization of a silylated peptide. We selected a short and low molecular building-block derived from the consensus collagen sequence [Pro-Hyp-Gly]. Interestingly, the sol–gel process occurs in physiological buffer, enabling the embedment of stem cells. This collagen-inspired hydrogel provides a cell-friendly environment comparable to natural collagen substrates, demonstrating its potency as a biomimetic scaffold.

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.

Structural Insights into the Glycine Pair Motifs in Type III Collagen.

Human type III collagen has been suggested to play vital roles in a series of pathophysiological conditions. Sequence analysis among major fibril-forming collagens (types I, II, and III) revealed that Gly-Gly pairs are a distinct sequence feature in type III collagen. This motif occurs more than five times as often in type III compared to type I and II collagens. We used an integrated computational modeling and biophysical approach to analyze the glycine pair motifs to understand how they govern the structure of type III collagen at the molecular level. Triple helical peptides to model the regions of type III collagen containing GG motifs were used to analyze structural and thermodynamic effects of GG incorporation into the collagen sequence. We found that when amino acids adjacent to a GG motif are charged, the collagen adopts a more flexible, random conformation. The GG motif led to altered hydrogen bond patterns and decreased global melting temperatures of the triple helical peptides. The local entropic destabilization effect of the glycine pair helps explain the difference in the flexibility between types I and III collagen fibrils. This finding reveals potential physiological roles of type III collagen in regulating the mechanical properties of collagen fibrils and may enable the design of future collagen-like materials with tunable mechanical properties.

Bioengineered Collagens.

There is a great deal of interest in obtaining recombinant collagen as an alternative source of material for biomedical applications and as an approach for obtaining basic structural and biological information. However, application of recombinant technology to collagen presents challenges, most notably the need for post-translational hydroxylation of prolines for triple-helix stability. Full length recombinant human collagens have been successfully expressed in cell lines, yeast, and several plant systems, while collagen fragments have been expressed in E. coli. In addition, bacterial collagen-like proteins can be expressed in high yields in E. coli and easily manipulated to incorporate biologically active sequences from human collagens. These expression systems allow manipulation of biologically active sequences within collagen, which has furthered our understanding of the relationships between collagen sequences, structure and function. Here, recombinant studies on collagen interactions with cell receptors, extracellular matrix proteins, and matrix metalloproteinases are reviewed, and discussed in terms of their potential biomaterial and biomedical applications.

Consequences of Glycine Mutations in the Fibronectin-binding Sequence of Collagen

Collagen and fibronectin (Fn) are two key extracellular matrix proteins, which are known to interact and jointly shape matrix structure and function. Most proteins that interact with collagen bind only to the native triple-helical form, whereas Fn is unusual in binding strongly to denatured collagen and more weakly to native collagen. The consequences of replacing a Gly by Ser at each position in the required (Gly-Xaa-Yaa)6 Fn-binding sequence are probed here, using model peptides and a recombinant bacterial collagen system. Fluorescence polarization and solid-state assays indicated that Gly replacements at four sites within the Fn-binding sequence led to decreased Fn binding to denatured collagen. Molecular dynamics simulations showed these Gly replacements interfered with the interaction of a collagen β-strand with the β-sheet structure of Fn modules seen in the high resolution crystal structure. Whereas previous studies showed that Gly to Ser mutations within an integrin-binding site caused no major structural perturbations, mutations within the Fn-binding site caused the triple helix to become highly sensitive to trypsin digestion. This trypsin susceptibility is consistent with the significant local unfolding and loss of hydrogen bonding seen in molecular dynamics simulations. Protease sensitivity resulting from mutations in the Fn-binding sequence could lead to degradation of type I collagen, early embryonic lethality, and the scarcity of reported osteogenesis imperfecta mutations in this region.

Genetic stability of an Escherichia coli strain engineered to produce a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis

Abstract The development of therapeutic DNA vaccines capable of recovering immunological tolerance through the induction of both CD4 + CD25 + FoxP3 + regulatory and CD3 + CD8 + C28-suppressor T cells, and/or inhibition of both autoreactive CD4 + CD28+ type 1 T helper and autoantibody-producing B cells offers a promising new strategy for the treatment of rheumatoid arthritis. Previously, we developed pcDNA-CCOL2A1, a novel therapeutic DNA vaccine, which encodes the full-length chicken type II collagen sequence, and demonstrated that the efficacy of this vaccine for treating rheumatoid arthritis was comparable to that of the current “gold standard” treatment, methotrexate. In this study, we investigated the genetic stability of a strain engineered to produce the vaccine during continuous passage and long-term storage at different temperatures. By screening a panel of 12 strains, we identified a DH5α strain that exhibited high levels (12.30 ± 0.05 mg L−1) of pcDNA-CCOL2A1 production after 15 h cultivation, and subsequently utilized this strain to establish a three-tier cells bank for future studies. Continuous passage of this strain for 100 inoculation times demonstrated that a higher percentage (>95%) of cells maintained the plasmid when cultivated under selective pressure (ampicillin) than under nonselective conditions, suggesting that the presence of antibiotics in the medium prevents the loss of the pcDNA-CCOL2A1 plasmid. Meanwhile, restriction digestion and gene sequencing analyses demonstrated that the pcDNA-CCOL2A1 vector remained stable, and that the plasmid sequence was conserved during this period. Lastly, the DH5α pcDNA-CCOL2A1 strain exhibited a high plasmid preservation (>90%) and high levels of plasmid production (9.05mg L−1) after storage for 60 months at −80 °C. Furthermore the plasmid extracted from the DH5α pcDNA-CCOL2A1 strain after storage for 60 months at −80 °C was transfected to COS-7 cells, it can stably express the target protein chicken type II collagen. Conversely, this strain exhibited a complete loss of capability after 24 and 18 months storage at −20 °C and 4 °C, respectively. These findings will facilitate further pilot-scale testing, and even industrial-scale production, of the novel therapeutic vaccine pcDNA-CCOL2A1.

X-Linked and Autosomal Recessive Alport Syndrome: Pathogenic Variant Features and Further Genotype-Phenotype Correlations.

Alport syndrome results from mutations in the COL4A5 (X-linked) or COL4A3/COL4A4 (recessive) genes. This study examined 754 previously- unpublished variants in these genes from individuals referred for genetic testing in 12 accredited diagnostic laboratories worldwide, in addition to all published COL4A5, COL4A3 and COL4A4 variants in the LOVD databases. It also determined genotype-phenotype correlations for variants where clinical data were available. Individuals were referred for genetic testing where Alport syndrome was suspected clinically or on biopsy (renal failure, hearing loss, retinopathy, lamellated glomerular basement membrane), variant pathogenicity was assessed using currently-accepted criteria, and variants were examined for gene location, and age at renal failure onset. Results were compared using Fisher’s exact test (DNA Stata). Altogether 754 new DNA variants were identified, an increase of 25%, predominantly in people of European background. Of the 1168 COL4A5 variants, 504 (43%) were missense mutations, 273 (23%) splicing variants, 73 (6%) nonsense mutations, 169 (14%) short deletions and 76 (7%) complex or large deletions. Only 135 of the 432 Gly residues in the collagenous sequence were substituted (31%), which means that fewer than 10% of all possible variants have been identified. Both missense and nonsense mutations in COL4A5 were not randomly distributed but more common at the 70 CpG sequences (p<10−41 and p<0.001 respectively). Gly>Ala substitutions were underrepresented in all three genes (p< 0.0001) probably because of an association with a milder phenotype. The average age at end-stage renal failure was the same for all mutations in COL4A5 (24.4 ±7.8 years), COL4A3 (23.3 ± 9.3) and COL4A4 (25.4 ± 10.3) (COL4A5 and COL4A3, p = 0.45; COL4A5 and COL4A4, p = 0.55; COL4A3 and COL4A4, p = 0.41). For COL4A5, renal failure occurred sooner with non-missense than missense variants (p<0.01). For the COL4A3 and COL4A4 genes, age at renal failure occurred sooner with two non-missense variants (p = 0.08, and p = 0.01 respectively). Thus DNA variant characteristics that predict age at renal failure appeared to be the same for all three Alport genes. Founder mutations (with the pathogenic variant in at least 5 apparently- unrelated individuals) were not necessarily associated with a milder phenotype. This study illustrates the benefits when routine diagnostic laboratories share and analyse their data.

Mapping the Effect of Gly Mutations in Collagen on α2β1 Integrin Binding

The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site.

Finding Britain's last hunter-gatherers: A new biomolecular approach to ‘unidentifiable’ bone fragments utilising bone collagen

In the last decade, our knowledge of the transition from foraging, fishing, and hunting to agricultural food production has been transformed through the molecular analysis of human remains. In Britain, however, the lack of Late Mesolithic human remains has limited our understanding of this dietary transition. Here, we report the use of a novel strategy to analyse otherwise overlooked material to identify additional human remains from this period. ZooMS, a method which uses bone collagen sequences to determine species, was applied to unidentifiable bone fragments from 5th millennium deposits from the Late Mesolithic site of Cnoc Coig (Oronsay, Inner Hebrides) using an innovative new methodology. All samples bar one produced ZooMS results, with 14/20 bone fragments identified as human, and the remainder a mixture of pig and seal. 70% of bone fragments had sufficient collagen for stable isotope analyses, however none of three human bone fragments analysed had sufficient endogenous DNA. By conducting AMS dating and stable isotope analysis on this identified collagen, we provide new data that supports the view that the exploitation of marine resources partially overlapped with the earliest agricultural communities in Britain, and thus argues against the idea that forager lifeways in Britain were immediately replaced by agriculture c.4000 cal. BC. Unfortunately, we were unable to explore the genetic relationship between contemporaneous farmers and foragers. However, the more persistent bone protein could be used to identify species, determine date, and assess diet. This novel approach is widely applicable to other early prehistoric sites with fragmentary skeletal material.

A Rapid and Simple LC-MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather.

Identification of the animal source of leather is difficult using traditional methods, including microscopic observation and PCR. In the present study, a LC-MS method was developed for detecting interspecies differences in the amino acid sequence of type I collagen, which is a major component of leather, among six animals (cattle, horse, pig, sheep, goat, and deer). After a dechroming procedure and trypsin digestion, six tryptic peptides of type I collagen were monitored by LC-MS in multiple reaction monitoring mode for the animal source identification using the patterns of the presence or absence of the marker peptides. We analyzed commercial leathers from various production areas using this method, and found some leathers in which the commercial label disagreed with the identified animal source. Our method enabled rapid and simple leather certification and could be applied to other animals whether or not their collagen sequences are available in public databases.

Collagen structure: new tricks from a very old dog.

The main features of the triple helical structure of collagen were deduced in the mid-1950s from fibre X-ray diffraction of tendons. Yet, the resulting models only could offer an average description of the molecular conformation. A critical advance came about 20 years later with the chemical synthesis of sufficiently long and homogeneous peptides with collagen-like sequences. The availability of these collagen model peptides resulted in a large number of biochemical, crystallographic and NMR studies that have revolutionized our understanding of collagen structure. High-resolution crystal structures from collagen model peptides have provided a wealth of data on collagen conformational variability, interaction with water, collagen stability or the effects of interruptions. Furthermore, a large increase in the number of structures of collagen model peptides in complex with domains from receptors or collagen-binding proteins has shed light on the mechanisms of collagen recognition. In recent years, collagen biochemistry has escaped the boundaries of natural collagen sequences. Detailed knowledge of collagen structure has opened the field for protein engineers who have used chemical biology approaches to produce hyperstable collagens with unnatural residues, rationally designed collagen heterotrimers, self-assembling collagen peptides, etc. This review summarizes our current understanding of the structure of the collagen triple helical domain (COL×3) and gives an overview of some of the new developments in collagen molecular engineering aiming to produce novel collagen-based materials with superior properties.

Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.

Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of publicly available collagen sequences improves, the simplicity of the PMF approach and suitable range of peptide sequence variation observed makes it the ideal method for initial taxonomic identification prior to further analysis by LC-based methods only when required.

Lysyl oxidase is associated with increased thrombosis and platelet reactivity

AbstractLysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Loxtg/tg) were generated. Pf4-Loxtg/tg mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Loxtg/tg platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Loxtg/tg platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors

Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å(2) of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition.

Trigger sequence can influence final morphology in the self-assembly of asymmetric telechelic polymers.

We report on a numerical study of polymer network formation of asymmetric biomimetic telechelic polymers with two reactive ends based on a self-assembling collagen, elastin or silk-like polypeptide sequence. The two reactive ends of the polymer can be activated independently using physicochemical triggers such as temperature and pH. We show, using a simple coarse grained model that the order in which this triggering occurs influences the final morphology. For both of collagen-silk and elastin-silk topologies we find that for relatively short connector chains the morphology of the assembly is greatly influenced by the order of the trigger, whereas for longer chains the equilibrium situation is more easily achieved. Moreover, self-assembly is greatly enhanced at moderate collagen interaction strength, due to facilitated binding and unbinding of the peptides. This finding indicates that both the trigger sequence and strength can be used to steer self-assembly in these biomimetic polymer systems.

Paper-based FRET for the direct detection of collagen triple helix.

In order to develop improved diagnosis and therapies for the highly prevalent chronic fibroproliferative diseases, it is of utmost importance to construct efficient assays to detect collagen biomarkers. We have successfully created a paper-based FRET assay for the fast, inexpensive and direct detection of collagen triple helix. We have demonstrated that the adsorption of the dye-labeled probe peptide onto the GO-immobilized paper quenches the fluorescence of the dye, while the hybridization of the probe peptide with the target collagen peptide results in the desorption of the probe peptide from GO, thus restoring the fluorescence. We have also shown that this novel assay is highly specific to the target collagen peptide sequence with little interference from other proteins, and it can be applied for quantitative detection in complex biological fluids. Our integration of the GO-based FRET assay with a patterned paper provides a powerful new tool for the detection of collagen molecules with many superior features: tiny volumes of samples, multichannel detection mode, easy operation and low-cost equipment. This assay may have promising applications to fast screen multiple target collagen sequences for the discovery of new collagen biomarkers.