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Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide
Author’s Accepted Manuscript Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide Linge Nian, Yue Hu, Caihong Fu, Chen Song, Jie Wang, Jianxi Xiao www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(17)30864-0 http://dx.doi.org/10.1016/j.talanta.2017.08.042 TAL17838
To appear in: Talanta Received date: 17 May 2017 Revised date: 27 July 2017 Accepted date: 11 August 2017 Cite this article as: Linge Nian, Yue Hu, Caihong Fu, Chen Song, Jie Wang and Jianxi Xiao, Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.08.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide
Linge Niana,1, Yue Hua,1, Caihong Fua, Chen Songa, Jie Wangb, Jianxi Xiaoa,b*
a
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and
Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China b
Key laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China
*
Corresponding author. [email protected]
ABSTRACT:
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.
1
Equal contribution. 1
Graphical abstract
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.
2
Keywords:
Collagen, fluorescence, self-quenching, peptide, targeting
1. Introduction
Collagen, as the most abundant protein in mammals, is widely found in various connective tissues, such as skin, bone, tendon, and ligament [1,2]. Collagen forms a molecular scaffold for mechanical strength and structural integrity of human body. Twenty-eight different types of collagen have been identified, including homotrimeric type II and type III collagen, and heterotrimeric type I and type IV collagen [3,4]. All kinds of collagen display a characteristic repetitive (Gly-X-Y)n amino acid sequence pattern, leading to the distinct triple helix structure [5,6].The close packing of three chains requires Gly to be every third residue, while residues in the X and Y positions are frequently Pro and Hyp, respectively [7]. (Gly-Pro-Hyp)n is considered as the most stabilizing sequence of collagen triple helix structure [8,9]. A variety of collagen fragments produced by collagen degradation serve as critical targets for novel diagnosis and therapies [10,11,12,13,14,15]. Excessive cleavage of collagen by proteolytic enzymes is involved in various diseases such as arthritis, chronic wounds and tumor [16,17]. Two fragments of type I and III collagen (C1M and C3M) were found to be associated with the extent of fibrosis in liver, while a fragment of type II collagen (C2M) was identified as a sensitive marker for cartilage erosion in arthritis [10,11]. The high level of serum C1M was recently discovered to be closely correlated with the increased mortality of postmenopausal women by a large prospective cohort study [12]. The detection of the degraded products of collagen has thus played an essential role for prognostic and therapeutic decisions in clinical practice. The development of effective antibodies and peptides to recognize these degraded products of collagen has received extensive attention [18,19,20,21,22,23,24,25]. A humanized monoclonal IgG1 antibody (D93) has been found to identify denatured collagen type IV by binding to a cryptic site of MMP-processed collagen [20]. Monoclonal antibodies E1E5 and E4A11 have been discovered to specifically interact with denatured collagen type II using ELISA-based screening [26].The peptide sequence TLTYTWS was discovered to specifically bind to a cryptic site on MMP-2 processed type IV collagen by phage display library screening [19]. These methods often suffer from tedious and time-consuming procedures, low specificity and binding affinity [19,20]. Furthermore, it remains technically challenging to discover novel antibodies for collagen due to its repetitive (Gly-X-Y)n amino acid sequences [20]. Therefore, it is still in urgent need to develop efficient assays for denatured collagen fragments, and one essential question is to discover probe peptides or antibodies that can 3
specifically recognize target collagen sequences. Fluorescence self-quenching has provided a powerful tool to investigate protein folding and conformational fluctuations as well as peptide aggregation [27,28,29]. We have recently constructed a novel fluorescence self-quenching assay to determine the helical composition of heterotrimeric collagen mimic peptides [30]. Herein we further develop the fluorescence self-quenching assay as a convenient tool to screen the targeting capability of a series of fluorescent probe peptides of variable lengths. We are able to discover a short probe peptide, and have demonstrated its power to specifically detect the desired collagen fragment in complex biological media by using fluorescence self-quenching assay.
2. Materials and methods
2.1. Materials Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp(tBu)-OH, 2-chlorotrityl chloride resin, hydroxybenzotriazole (HOBt), and O-(Benzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Jier Biochemical Company (Shanghai, China); Diisopropylethylamine (DIEA) was purchased from Hanhong Chemical Technology Co., Ltd (Shanghai, China); 5(6)-Carboxytetramethylrhodamine (FAM) was provided by Aladdin Industrial Corporation (Shanghai, China); trifluoroacetic acid (TFA) was purchased from J&K (Shanghai, China); Triisopropylsilane (Tis) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan); BSA (Albumin bovine V), hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase were obtained from Sangon Biotech Co., Ltd (Shanghai, China). All the commercial reagents were of analytical grade and were used without further purification. Ultrapure water was used to prepare all the solutions.
2.2. Peptide synthesis Peptide G(POG)10 was synthesized by Chinese Peptide Company (Hangzhou, China). Other peptides FAM-G(POG)10, FAM-G(POG)8, FAM-G(POG)6, FAM-G(POG)4, G(POG)8, G(POG)6 and G(POG)4 were synthesized in-house by standard Fmoc solid phase synthesis method using 2-chlorotrityl chloride resin. Stepwise couplings of amino acids were carried out using Fmoc-amino acids (4 eq.), DIEA (6 eq.), HBTU (4 eq.) and HOBt (4 eq.). After each step of coupling, the reaction mixture was washed with DMF and DCM three times, and 20% piperidine in DMF was applied to eliminate the Fmoc protection group. Test reagent (2% ethanol DMF, 2% chloranil DMF) was added to check the status of coupling reaction and Fmoc deprotetction. FAM was conjugated to the N-terminal of the peptide by using FAM (12 eq.), DIEA (6 eq.) and activator reagent 4
PyAOP (12 eq.). TFA/TIS/H2O (95:2.5:2.5) was employed to cleave the peptides from the resin and eliminate the tBu groups. The peptides were collected by precipitation with cold Et2O, and crude products were obtained after re-suspension in cold Et2O, sonication and centrifugation. These peptides were purified using reverse phase HPLC on a C18 column, and the purity of the peptides were confirmed by mass spectrometry. m/z calculated 3127.4 [M+Na] + for FAM-G(POG)10, found 3127.8 [M+Na]+; m/z calculated 2592.8 [M+Na]+ for FAM-G(POG)8, found 2593.2 [M+Na]+; m/z calculated 2058.2 [M+Na]+ for FAM-G(POG)6, found 2059.0 [M+Na]+; m/z calculated 1523.6 [M+Na]+ for FAM-G(POG)4, found 1524.8 ; m/z calculated 2234.5 [M+Na]+ for G(POG)8, found 2233.7 [M+Na]+; m/z calculated 1678.9 [M]+ for G(POG)6, found 1678.7 [M]+; m/z calculated 1144.3 [M]+ for G(POG)4, found 1144.5 [M]+.
2.3.
Fluorescence Assays Fluorescence spectra were recorded on a RF-5301PC fluorescence spectrometer using a Xenon lamp as an
excitation source (Shimadzu Corporation, Kyoto, Japan). For each fluorescent probe peptide A, B, C, and D, three solutions with a concentration of 300 µM were prepared in 20 mM PBS buffer at pH 7.4, and were incubated at 4oC for >24 hrs to ensure the homotrimer formation. One peptide solution was kept at 4 oC , while the second solution was heated at 90oC for 25 min, and the third solution was re-equilibrated at 4oC for 48 hrs after the heating at 90oC for 25 min. The emission spectra were recorded for each peptide under all these three conditions immediately after diluting the peptide solutions to a final concentration of 1 µM. Fluorescence measurements were conducted from 505 to 650 nm with a 1 nm increment per step at an excitation wavelength of 493 nm. In order to evaluate the possible heterotrimer formation between the fluorescent probe peptides and the target peptides, the peptide mixtures of AA’, AB’, AC’, AD’, BA’, BB’, BC’, BD’, CA’, CB’, CC’, and CD’ were prepared, with a concentration of 300 µM for probe peptide A, B and C, and 600 µM for target peptide A’, B’, C’, and D’ in 20 mM PBS buffer at pH 7.4. Three solutions of each peptide mixture were prepared and incubated at 4oC for >24 hrs. One solution of the peptide mixture was kept at 4 oC, while the second solution was heated at 90oC for 25 min, and the third solution was re-equilibrated at 4oC for 48 hrs after the heating at 90oC for 25 min. The emission spectra were recorded for each peptide mixture under all these three conditions immediately after diluting the solutions to a final concentration of 1 µM for probe peptide. For the quantitative assay, different final concentrations of the target peptide A’ (0, 100, 200, 300, 400, 500, and 1000 nM) were prepared in the mixture with probe peptides B (1 µM) and A (1 µM), respectively. Urine and saliva samples were obtained from laboratory personnel. The samples were centrifuged at 5000 rpm for 5 minutes and diluted to 20% by 20 mM PBS buffer (pH 7.4). The BA’ and AA’ mixtures with different final concentrations of the target peptide A’ (0, 100, 200, 400,600, 800, and 1000 nM) were added in the biological media, and the 5
fluorescence spectra were recorded. Proteins BSA (Bovine serum albumin), hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase with a final concentration of 2 µM were added to the prepared solution of peptide B and the BA’ mixture, respectively, to investigate the interference of other proteins in the detection of the target peptide A’. All measurements were repeated 3 times.
3. Results and discussion 3.1. Design of the fluorescent collagen peptide probe In order to identify a short effective peptide probe that can specifically recognize the target collagen sequences, we have constructed a series of probe peptides of various lengths of (Pro-Hyp-Gly)n triplets (n=4, 6, 8, 10), which are labeled with widely used fluorescein-based fluorescent dye (FAM) (Table 1). Peptide FAM-G(PRGPOG)5 was used as the probe in our previous report of fluorescence self-quenching assay to determine the helical composition of heterotrimeric collagen mimic peptides [30]. In order to discover short probe peptides, the sequence (POG)n is chosen here since (POG)n is suggested to be the most stabilizing collagen sequence for the triple helix structure [8]. The target collagen sequences are also designed with different lengths of (Pro-Hyp-Gly)n triplets (n=4, 6, 8, 10) without any fluorescent labels , since Pro-Hyp-Gly is the most populous triplet in collagen (Table 1). We herein construct a fluorescence self-quenching assay to screen the capability of various probe peptides to recognize the target collagen sequences (Scheme 1). We hypothesize that if FAM-labeled probe peptide has matched length, and can recognize the unlabeled target peptide, they will strongly bind with each other to form a heterotrimer, which possesses strong fluorescence. However, if FAM-labeled probe peptide has mismatched length, and cannot recognize the target peptide, the probe peptide will form homotrimer by itself, leading to the fluorescence self-quenching (Scheme 1). The difference in fluorescence intensity of the peptide mixture will allow us to facilely evaluate if a probe peptide can bind with the target peptide.
3.2. Homotrimer formation of the probe peptides In our hypothesis of the fluorescence self-quenching assay to investigate the binding capability of probe peptides, there is a prerequisite for the probe peptides to form homotrimers. We therefore first evaluate if the probe peptide can form homotrimer by itself (Figure 1). Fluorescence profiles are measured for solutions of peptide FAM-G(POG)10 incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red) (Figure 1a). FAM-G(POG)10 is in the homotrimer state at 4oC, therefore displaying relatively low fluorescence due to the close packing of the three chains resulting in fluorescence self-quenching of the fluorescence dye. When FAM-G(POG)10 is heated to 90oC, it is in the monomer state and its fluorescence is 6
significantly increased. When FAM-G(POG)10 is re-equilibrated at 4oC, its fluorescence is reduced to the original value at 4oC, indicating the reformation of homotrimer (Figure 1a). The fluorescence profiles of FAM-G(POG)10 at these three conditions clearly demonstrate that this probe peptide shows a strong capability to form homotrimer. Compared with FAM-G(POG)10, peptides FAM-G(POG)8 and FAM-G(POG)6 show almost the same temperature-dependent fluorescence profiles: high fluorescence at 90oC as well as comparatively low fluorescence at originally 4oC or re-equilibrated back to 4oC (Figure 1b-c). These results indicate that both FAM-G(POG)8 and FAM-G(POG)6 can form homotrimers, though FAM-G(POG)6 may display a slightly weaker capability since it cannot completely come back to the original value after re-equilibration. Most notably, peptide FAM-G(POG)4 shows a totally different fluorescence profile that the fluorescence spectra at those three conditions are almost overlapped (Figure 1d). The strong fluorescence at 4 oC as well as 90oC indicates that FAM-G(POG)4 remains in the monomer state at 4oC. In summary, FAM-G(POG)4 seems too short to form homotrimer structure, while longer peptides FAM-G(POG)10, FAM-G(POG)8 and FAM-G(POG)6 can form homotrimers. These three peptides are possible candidates for our fluorescence self-quenching assay, and their capability to recognize target collagen sequences will be further evaluated .
3.3. Targeting capability of probe peptide FAM-G(POG)10 We first examine the capability of the probe peptide FAM-G(POG)10 (denoted as peptide A) to interact with target collagen sequences of various lengths. Fluorescence profiles of the mixture of probe peptide FAM-G(POG)10 with various target peptides G(POG)10, G(POG)8, G(POG)6 and G(POG)4 (denoted as peptide A’, B’, C’ and D’, respectively) are recorded (Figure 2). The peptide mixture AA’ shows a relatively weak fluorescence at 4oC, suggesting that both peptides form homotrimers by themselves; while the fluorescence of AA’ becomes significantly increased at 90oC, indicating the transition into monomer states for both peptides. After the re-equilibration at 4oC, the fluorescence of AA’ remains much stronger than the original fluorescence at 4oC, suggesting that peptide mixture AA’ forms heterotrimer instead of returning back to the homotrimer state (Figure 2a). The distinct fluorescence profiles under these three conditions have demonstrated that probe peptide A can recognize target sequence A’, and form heterotrimer. The peptide mixture AB’ shows almost the same temperature-dependent fluorescence profiles as AA’ (Figure 2b). Particularly, the fluorescence of AB’ after the re-equilibration at 4oC is much stronger than the original fluorescence at 4oC, suggesting the heterotrimer formation between A and B’. In contrast, peptide mixtures AC’ and AD’ show quite different fluorescence profiles (Figure 2c-d). They also display similar weak fluorescence at original 4oC as well as strong fluorescence at 90oC; however, their fluorescence after re-equilibration is only a little higher than, or equal to the original fluorescence at 4 oC , suggesting that there are 7
very weak interaction between A and C’, and even no significant interaction between A and D’. In summary, the interactions between probe peptide A and target peptides A’, B’, C’ and D’ are length-dependent. As the target peptide becomes shorter, the binding between the probe and target peptides becomes much weaker.
3.4. Targeting capability of probe peptide FAM-G(POG)8 The capability of the probe peptide FAM-G(POG)8 (denoted as peptide B) to interact with target collagen sequences of various lengths are then evaluated. Fluorescence profiles of the mixture of probe peptide FAM-G(POG)8 with various target peptides G(POG)10, G(POG)8, G(POG)6, and G(POG)4 are recorded (Figure 3). The peptide mixtures BA’ and BB’ show very similar fluorescence profiles that their fluorescence after the re-equilibration at 4oC is much stronger than the original fluorescence at 4oC, suggesting that peptide B can strongly bind with A’ and B’ to form heterotrimers (Figure 3a-b). Meanwhile, the peptide mixtures BC’ and BD’ show very similar fluorescence profiles that their fluorescence after the re-equilibration at 4oC is almost the same as the original fluorescence at 4oC, suggesting that peptide B cannot form heterotrimers with C’ and D’ (Figure 3c-d). To conclude, probe peptide FAM-G(POG)8 also shows length-dependent interaction with target collagen sequences, and it displays as strong capability as FAM-G(POG)10 to bind with target collagen peptides G(POG)10 and G(POG)8.
3.5. Targeting capability of probe peptide FAM-G(POG)6 The interactions of the probe peptide FAM-G(POG)6 (denoted as peptide C) with target collagen sequences of various lengths are also investigated by comparing the fluorescence spectra of the peptide mixtures (CA’, CB’, CC’, and CD’) (Figure 4). The peptide mixtures CA’ and CB’ show a little stronger fluorescence after the re-equilibration than the original fluorescence at 4oC , suggesting a weak interaction of peptide C with A’ and B’ (Figure 4a-b). In contrast, the peptide mixtures CC’ and CD’ show very similar fluorescence after the re-equilibration at 4oC as compared to the original fluorescence at 4oC, indicating the absence of significant interactions between peptide C and peptide C’ or D’ (Figure 4c-d). Compared with FAM-G(POG)10 and FAM-G(POG)8, probe peptide FAM-G(POG)6 does not display strong interaction with target collagen peptides including G(POG)10 and G(POG)8.
3.6. Linear detection of target collage peptides by probe FAM-G(POG)8 Among the four constructed probe peptides, only FAM-G(POG)10 and FAM-G(POG)8 form homotrimers by themselves as well as heterotrimers when hybridized with target collagen sequences G(POG) 10 and G(POG)8. 8
FAM-G(POG)8 is shorter than FAM-G(POG)10, therefore it is chosen as the appropriate probe peptide to further develop a quantitative assay for target collagen sequence G(POG)10. The effect of pH and salt on the fluorescence characterization of probe peptide FAM-G(POG)8 has been first evaluated (Figure S1a). The fluorescence intensity of the probe peptide is similar at pH 7.4 and pH 8.0, while it is largely reduced at acidic pH 6.0. Compared with pH, various salt concentrations (0, 150 mM and 300 mM) result in much smaller changes in fluorescence. The fluorescence restoration capability of the target peptide G(POG) 10 is further investigated under different pH and salt conditions (Figure S1b). The recovery efficiency of G(POG) 10 on the FAM-G(POG)8 is much smaller at pH 6 than pH 7.4 and pH 8, while the presence of extra salt does not significantly affect the fluorescence. pH 7.4 PBS buffer without any salt is thus chosen as the condition for further characterization. When FAM-G(POG)8 is hybridized with various concentrations of G(POG) 10, a proportional relationship between the fluorescence of the peptide system and the concentration of the target peptide is found (R2 = 0.99) (Figure 5a). The system shows a linear range from 100 to 1000 nM, with an accurate determination of the target collagen sequence as low as 84 nM, which is comparable to the FAM-G(POG)10-based fluorescence assay with a linear range of 100-1000 nM and a detection limit of 55 nM (Figure S2a). Theses results demonstrate that the shorter peptide FAM-G(POG)8 provides a capable probe for the detection of target collagen peptide.
3.7. Detection of target collagen sequences in biologic fluids The fluorescence self-quenching assay together with the probe peptide FAM-G(POG)8 is further applied to detect target collagen sequences in human urine samples (Figure 5b). A linear relationship between the fluorescence of the peptide system and the concentration of target collagen sequence G(POG)10 is observed in the range of 100-1000 nM (R2 = 0.98) with a detection limit of 74 nM (Figure 5b). A similar proportional relationship between the fluorescence of the system and the concentration of target peptide is also identified in human saliva samples (R2 = 0.97) (Figure 5c). The FAM-G(POG)8-based fluorescence self-quenching assay has reached similar linear ranges and detection limits as the FAM-G(POG)10-based method (Figure S2b-c). The presence of target peptide G(POG)10 in human urine and saliva samples is also confirmed by Mass spectroscopy (Figure S3). These results have demonstrated that probe peptide FAM-G(POG)8 is capable to detect target collagen peptides in complex biological media. The applicability of this FAM-G(POG)8-based fluorescence self-quenching assay is evaluated in urine and saliva samples fortified with different concentrations of target peptide G(POG) 10 (Table S1). Analysis of the results of spiked samples displays the recoveries within the range of 98.7% - 108.4%, demonstrating the accuracy of the method and its suitability for routine analysis. These recoveries using the probe peptide FAM-G(POG)8 are comparable to those obtained by the assay using the probe peptide FAM-G(POG)10 (100.1% 9
- 107.9%) (Table S1). It suggests that the short peptide FAM-G(POG)8 is an excellent probe for the fluorescence self-quenching assay in the detection of target collagen peptide in biological media.
3.8. Interference The interference of other proteins in the detection of target collagen peptide sequence G(POG) 10 using probe peptide FAM-G(POG)8 is examined (Figure 6). The fluorescence intensities are measured for the probe peptide FAM-G(POG)8 in the absence and presence of nonspecific proteins BSA, hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase, respectively (purple bar). They all show relatively similar fluorescence intensities, indicating that these nonspecific proteins do not interfere with the fluorescence characterization of the probe peptide. Compared with the target peptide G(POG) 10, these proteins all exhibit much less florescence restoration capability on the FAM-G(POG)8, indicating the highly selective recognition of target collagen sequence by the probe peptide. Meanwhile, the fluorescence intensities are recorded for FAM-G(POG)8 hybridized with G(POG)10 at 518 nm in the absence and presence of nonspecific proteins (red bar). The addition of extra proteins does not significantly affect the fluorescence intensity of the hybridized peptides, indicating that the probe peptide FAM-G(POG)8 is highly specific for the target collagen sequence (Figure 6). FAM-G(POG)8 may provide a sensitive and selective probe for the detection of target collagen peptide with little interference from other proteins.
4. Conclusions As the major structural protein of human body, the degradation of collagen is involved in various diseases such as arthritis, chronic wounds and tumor [16,24]. The construction of effective assays for the detection of collagen fragments plays critical roles in diagnostic, prognostic and therapeutic decisions in collagen-related diseases. An essential question for the assay development is to discover novel 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 examined the fluorescence profiles of the probe peptides (FAM-G(POG)10, FAM-G(POG)8, FAM-G(POG)6 and FAM-G(POG)4) hybridized with various target collagen sequences, and revealed that the targeting capability of probe peptides is length-dependent. As the probe peptides become shorter, they show weaker ability to bind with the target peptides. It is observed that only FAM-G(POG)8 and peptides with higher lengths can specifically recognize target peptides. Short peptides have several advantages such as easy synthesis 10
and modification. We have further demonstrated that this newly discovered short probe peptide FAM-G(POG)8 can be applied to specifically detect the desired collagen fragment in complex biological media. The FAM-G(POG)8-based fluorescence self-quenching assay proves to be very selective for target collagen sequences with little interference from other proteins. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers. It may have very promising applications in the discovery of potent probe peptides for various protein biomarkers involved in pathological conditions.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21305056), the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2016-k10) and open fund of State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (Grant No. T151402).
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Scheme 1. Schematic Illustration of monitoring the interaction of target peptides and fluorescent probe peptides of different lengths using fluorescence self-quenching assay.
Figure 1. Fluorescence characterization of the FAM-labeled probe peptides: FAM-G(POG)10 (a), FAM-G(POG)8 (b), FAM-G(POG)6 (c), and FAM-G(POG)4 (d). Fluorescence profiles are recorded for the peptide solutions incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 2. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)10 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 3. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)8 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 4. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)6 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red).
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Figure 5. The proportional fluorescence restoration of probe peptide FAM-G(POG)8 by the hybridization with target collagen peptide sequence G(POG)10 in PBS (a), human urine (b) and saliva (c) samples. The fluorescence emission spectra are recorded for peptide FAM-G(POG)8 in the presence of various concentrations of G(POG) 10 (0, 100, 200, 400, 600, 800,1000 nM). The fluorescence intensity monitored at 518 nm is plotted as a function of the concentration of G(POG)10. Figure 6. The interference of other proteins in the detection of target collagen peptide sequence G(POG) 10 using probe peptide FAM-G(POG)8. The fluorescence intensities are measured for probe peptide FAM-G(POG)8 at 518 nm in the absence and presence of nonspecific proteins BSA, hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase, respectively (purple). The fluorescence restoration of probe peptide FAM-G(POG)8 by the target peptide G(POG)10 is determined in the absence and presence of the nonspecific proteins (red).
Table 1. Design of FAM-labeled probe peptides and unlabeled target peptides with different lengths. Peptide Name
Sequence
A
FAM-G(GPO)10
B
FAM-G(GPO)8
C
FAM-G(GPO)6
D
FAM-G(GPO)4
A’
G(GPO)10
B’
G(GPO)8
C’
G(GPO)6
D’
G(GPO)4
Function
Probe
Target
14
Figure 1
Figure 2
15
Figure 3
Figure 4
16
Figure 5
Figure 6
17
Highlights:
The development of novel assays to detect collagen fragments is of utmost importance.
Fluorescence self-quenching assay has been established to detect collagen sequences.
The targeting ability of the probe peptides has been found to be length-dependent.
A short probe peptide has been discovered to specifically recognize target peptides.
The assay provides a powerful new tool to identify probe peptides for various biomarkers.
18
PII: DOI: Reference:
S0039-9140(17)30864-0 http://dx.doi.org/10.1016/j.talanta.2017.08.042 TAL17838
To appear in: Talanta Received date: 17 May 2017 Revised date: 27 July 2017 Accepted date: 11 August 2017 Cite this article as: Linge Nian, Yue Hu, Caihong Fu, Chen Song, Jie Wang and Jianxi Xiao, Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.08.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide
Linge Niana,1, Yue Hua,1, Caihong Fua, Chen Songa, Jie Wangb, Jianxi Xiaoa,b*
a
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and
Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China b
Key laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China
*
Corresponding author. [email protected]
ABSTRACT:
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.
1
Equal contribution. 1
Graphical abstract
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.
2
Keywords:
Collagen, fluorescence, self-quenching, peptide, targeting
1. Introduction
Collagen, as the most abundant protein in mammals, is widely found in various connective tissues, such as skin, bone, tendon, and ligament [1,2]. Collagen forms a molecular scaffold for mechanical strength and structural integrity of human body. Twenty-eight different types of collagen have been identified, including homotrimeric type II and type III collagen, and heterotrimeric type I and type IV collagen [3,4]. All kinds of collagen display a characteristic repetitive (Gly-X-Y)n amino acid sequence pattern, leading to the distinct triple helix structure [5,6].The close packing of three chains requires Gly to be every third residue, while residues in the X and Y positions are frequently Pro and Hyp, respectively [7]. (Gly-Pro-Hyp)n is considered as the most stabilizing sequence of collagen triple helix structure [8,9]. A variety of collagen fragments produced by collagen degradation serve as critical targets for novel diagnosis and therapies [10,11,12,13,14,15]. Excessive cleavage of collagen by proteolytic enzymes is involved in various diseases such as arthritis, chronic wounds and tumor [16,17]. Two fragments of type I and III collagen (C1M and C3M) were found to be associated with the extent of fibrosis in liver, while a fragment of type II collagen (C2M) was identified as a sensitive marker for cartilage erosion in arthritis [10,11]. The high level of serum C1M was recently discovered to be closely correlated with the increased mortality of postmenopausal women by a large prospective cohort study [12]. The detection of the degraded products of collagen has thus played an essential role for prognostic and therapeutic decisions in clinical practice. The development of effective antibodies and peptides to recognize these degraded products of collagen has received extensive attention [18,19,20,21,22,23,24,25]. A humanized monoclonal IgG1 antibody (D93) has been found to identify denatured collagen type IV by binding to a cryptic site of MMP-processed collagen [20]. Monoclonal antibodies E1E5 and E4A11 have been discovered to specifically interact with denatured collagen type II using ELISA-based screening [26].The peptide sequence TLTYTWS was discovered to specifically bind to a cryptic site on MMP-2 processed type IV collagen by phage display library screening [19]. These methods often suffer from tedious and time-consuming procedures, low specificity and binding affinity [19,20]. Furthermore, it remains technically challenging to discover novel antibodies for collagen due to its repetitive (Gly-X-Y)n amino acid sequences [20]. Therefore, it is still in urgent need to develop efficient assays for denatured collagen fragments, and one essential question is to discover probe peptides or antibodies that can 3
specifically recognize target collagen sequences. Fluorescence self-quenching has provided a powerful tool to investigate protein folding and conformational fluctuations as well as peptide aggregation [27,28,29]. We have recently constructed a novel fluorescence self-quenching assay to determine the helical composition of heterotrimeric collagen mimic peptides [30]. Herein we further develop the fluorescence self-quenching assay as a convenient tool to screen the targeting capability of a series of fluorescent probe peptides of variable lengths. We are able to discover a short probe peptide, and have demonstrated its power to specifically detect the desired collagen fragment in complex biological media by using fluorescence self-quenching assay.
2. Materials and methods
2.1. Materials Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp(tBu)-OH, 2-chlorotrityl chloride resin, hydroxybenzotriazole (HOBt), and O-(Benzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Jier Biochemical Company (Shanghai, China); Diisopropylethylamine (DIEA) was purchased from Hanhong Chemical Technology Co., Ltd (Shanghai, China); 5(6)-Carboxytetramethylrhodamine (FAM) was provided by Aladdin Industrial Corporation (Shanghai, China); trifluoroacetic acid (TFA) was purchased from J&K (Shanghai, China); Triisopropylsilane (Tis) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan); BSA (Albumin bovine V), hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase were obtained from Sangon Biotech Co., Ltd (Shanghai, China). All the commercial reagents were of analytical grade and were used without further purification. Ultrapure water was used to prepare all the solutions.
2.2. Peptide synthesis Peptide G(POG)10 was synthesized by Chinese Peptide Company (Hangzhou, China). Other peptides FAM-G(POG)10, FAM-G(POG)8, FAM-G(POG)6, FAM-G(POG)4, G(POG)8, G(POG)6 and G(POG)4 were synthesized in-house by standard Fmoc solid phase synthesis method using 2-chlorotrityl chloride resin. Stepwise couplings of amino acids were carried out using Fmoc-amino acids (4 eq.), DIEA (6 eq.), HBTU (4 eq.) and HOBt (4 eq.). After each step of coupling, the reaction mixture was washed with DMF and DCM three times, and 20% piperidine in DMF was applied to eliminate the Fmoc protection group. Test reagent (2% ethanol DMF, 2% chloranil DMF) was added to check the status of coupling reaction and Fmoc deprotetction. FAM was conjugated to the N-terminal of the peptide by using FAM (12 eq.), DIEA (6 eq.) and activator reagent 4
PyAOP (12 eq.). TFA/TIS/H2O (95:2.5:2.5) was employed to cleave the peptides from the resin and eliminate the tBu groups. The peptides were collected by precipitation with cold Et2O, and crude products were obtained after re-suspension in cold Et2O, sonication and centrifugation. These peptides were purified using reverse phase HPLC on a C18 column, and the purity of the peptides were confirmed by mass spectrometry. m/z calculated 3127.4 [M+Na] + for FAM-G(POG)10, found 3127.8 [M+Na]+; m/z calculated 2592.8 [M+Na]+ for FAM-G(POG)8, found 2593.2 [M+Na]+; m/z calculated 2058.2 [M+Na]+ for FAM-G(POG)6, found 2059.0 [M+Na]+; m/z calculated 1523.6 [M+Na]+ for FAM-G(POG)4, found 1524.8 ; m/z calculated 2234.5 [M+Na]+ for G(POG)8, found 2233.7 [M+Na]+; m/z calculated 1678.9 [M]+ for G(POG)6, found 1678.7 [M]+; m/z calculated 1144.3 [M]+ for G(POG)4, found 1144.5 [M]+.
2.3.
Fluorescence Assays Fluorescence spectra were recorded on a RF-5301PC fluorescence spectrometer using a Xenon lamp as an
excitation source (Shimadzu Corporation, Kyoto, Japan). For each fluorescent probe peptide A, B, C, and D, three solutions with a concentration of 300 µM were prepared in 20 mM PBS buffer at pH 7.4, and were incubated at 4oC for >24 hrs to ensure the homotrimer formation. One peptide solution was kept at 4 oC , while the second solution was heated at 90oC for 25 min, and the third solution was re-equilibrated at 4oC for 48 hrs after the heating at 90oC for 25 min. The emission spectra were recorded for each peptide under all these three conditions immediately after diluting the peptide solutions to a final concentration of 1 µM. Fluorescence measurements were conducted from 505 to 650 nm with a 1 nm increment per step at an excitation wavelength of 493 nm. In order to evaluate the possible heterotrimer formation between the fluorescent probe peptides and the target peptides, the peptide mixtures of AA’, AB’, AC’, AD’, BA’, BB’, BC’, BD’, CA’, CB’, CC’, and CD’ were prepared, with a concentration of 300 µM for probe peptide A, B and C, and 600 µM for target peptide A’, B’, C’, and D’ in 20 mM PBS buffer at pH 7.4. Three solutions of each peptide mixture were prepared and incubated at 4oC for >24 hrs. One solution of the peptide mixture was kept at 4 oC, while the second solution was heated at 90oC for 25 min, and the third solution was re-equilibrated at 4oC for 48 hrs after the heating at 90oC for 25 min. The emission spectra were recorded for each peptide mixture under all these three conditions immediately after diluting the solutions to a final concentration of 1 µM for probe peptide. For the quantitative assay, different final concentrations of the target peptide A’ (0, 100, 200, 300, 400, 500, and 1000 nM) were prepared in the mixture with probe peptides B (1 µM) and A (1 µM), respectively. Urine and saliva samples were obtained from laboratory personnel. The samples were centrifuged at 5000 rpm for 5 minutes and diluted to 20% by 20 mM PBS buffer (pH 7.4). The BA’ and AA’ mixtures with different final concentrations of the target peptide A’ (0, 100, 200, 400,600, 800, and 1000 nM) were added in the biological media, and the 5
fluorescence spectra were recorded. Proteins BSA (Bovine serum albumin), hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase with a final concentration of 2 µM were added to the prepared solution of peptide B and the BA’ mixture, respectively, to investigate the interference of other proteins in the detection of the target peptide A’. All measurements were repeated 3 times.
3. Results and discussion 3.1. Design of the fluorescent collagen peptide probe In order to identify a short effective peptide probe that can specifically recognize the target collagen sequences, we have constructed a series of probe peptides of various lengths of (Pro-Hyp-Gly)n triplets (n=4, 6, 8, 10), which are labeled with widely used fluorescein-based fluorescent dye (FAM) (Table 1). Peptide FAM-G(PRGPOG)5 was used as the probe in our previous report of fluorescence self-quenching assay to determine the helical composition of heterotrimeric collagen mimic peptides [30]. In order to discover short probe peptides, the sequence (POG)n is chosen here since (POG)n is suggested to be the most stabilizing collagen sequence for the triple helix structure [8]. The target collagen sequences are also designed with different lengths of (Pro-Hyp-Gly)n triplets (n=4, 6, 8, 10) without any fluorescent labels , since Pro-Hyp-Gly is the most populous triplet in collagen (Table 1). We herein construct a fluorescence self-quenching assay to screen the capability of various probe peptides to recognize the target collagen sequences (Scheme 1). We hypothesize that if FAM-labeled probe peptide has matched length, and can recognize the unlabeled target peptide, they will strongly bind with each other to form a heterotrimer, which possesses strong fluorescence. However, if FAM-labeled probe peptide has mismatched length, and cannot recognize the target peptide, the probe peptide will form homotrimer by itself, leading to the fluorescence self-quenching (Scheme 1). The difference in fluorescence intensity of the peptide mixture will allow us to facilely evaluate if a probe peptide can bind with the target peptide.
3.2. Homotrimer formation of the probe peptides In our hypothesis of the fluorescence self-quenching assay to investigate the binding capability of probe peptides, there is a prerequisite for the probe peptides to form homotrimers. We therefore first evaluate if the probe peptide can form homotrimer by itself (Figure 1). Fluorescence profiles are measured for solutions of peptide FAM-G(POG)10 incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red) (Figure 1a). FAM-G(POG)10 is in the homotrimer state at 4oC, therefore displaying relatively low fluorescence due to the close packing of the three chains resulting in fluorescence self-quenching of the fluorescence dye. When FAM-G(POG)10 is heated to 90oC, it is in the monomer state and its fluorescence is 6
significantly increased. When FAM-G(POG)10 is re-equilibrated at 4oC, its fluorescence is reduced to the original value at 4oC, indicating the reformation of homotrimer (Figure 1a). The fluorescence profiles of FAM-G(POG)10 at these three conditions clearly demonstrate that this probe peptide shows a strong capability to form homotrimer. Compared with FAM-G(POG)10, peptides FAM-G(POG)8 and FAM-G(POG)6 show almost the same temperature-dependent fluorescence profiles: high fluorescence at 90oC as well as comparatively low fluorescence at originally 4oC or re-equilibrated back to 4oC (Figure 1b-c). These results indicate that both FAM-G(POG)8 and FAM-G(POG)6 can form homotrimers, though FAM-G(POG)6 may display a slightly weaker capability since it cannot completely come back to the original value after re-equilibration. Most notably, peptide FAM-G(POG)4 shows a totally different fluorescence profile that the fluorescence spectra at those three conditions are almost overlapped (Figure 1d). The strong fluorescence at 4 oC as well as 90oC indicates that FAM-G(POG)4 remains in the monomer state at 4oC. In summary, FAM-G(POG)4 seems too short to form homotrimer structure, while longer peptides FAM-G(POG)10, FAM-G(POG)8 and FAM-G(POG)6 can form homotrimers. These three peptides are possible candidates for our fluorescence self-quenching assay, and their capability to recognize target collagen sequences will be further evaluated .
3.3. Targeting capability of probe peptide FAM-G(POG)10 We first examine the capability of the probe peptide FAM-G(POG)10 (denoted as peptide A) to interact with target collagen sequences of various lengths. Fluorescence profiles of the mixture of probe peptide FAM-G(POG)10 with various target peptides G(POG)10, G(POG)8, G(POG)6 and G(POG)4 (denoted as peptide A’, B’, C’ and D’, respectively) are recorded (Figure 2). The peptide mixture AA’ shows a relatively weak fluorescence at 4oC, suggesting that both peptides form homotrimers by themselves; while the fluorescence of AA’ becomes significantly increased at 90oC, indicating the transition into monomer states for both peptides. After the re-equilibration at 4oC, the fluorescence of AA’ remains much stronger than the original fluorescence at 4oC, suggesting that peptide mixture AA’ forms heterotrimer instead of returning back to the homotrimer state (Figure 2a). The distinct fluorescence profiles under these three conditions have demonstrated that probe peptide A can recognize target sequence A’, and form heterotrimer. The peptide mixture AB’ shows almost the same temperature-dependent fluorescence profiles as AA’ (Figure 2b). Particularly, the fluorescence of AB’ after the re-equilibration at 4oC is much stronger than the original fluorescence at 4oC, suggesting the heterotrimer formation between A and B’. In contrast, peptide mixtures AC’ and AD’ show quite different fluorescence profiles (Figure 2c-d). They also display similar weak fluorescence at original 4oC as well as strong fluorescence at 90oC; however, their fluorescence after re-equilibration is only a little higher than, or equal to the original fluorescence at 4 oC , suggesting that there are 7
very weak interaction between A and C’, and even no significant interaction between A and D’. In summary, the interactions between probe peptide A and target peptides A’, B’, C’ and D’ are length-dependent. As the target peptide becomes shorter, the binding between the probe and target peptides becomes much weaker.
3.4. Targeting capability of probe peptide FAM-G(POG)8 The capability of the probe peptide FAM-G(POG)8 (denoted as peptide B) to interact with target collagen sequences of various lengths are then evaluated. Fluorescence profiles of the mixture of probe peptide FAM-G(POG)8 with various target peptides G(POG)10, G(POG)8, G(POG)6, and G(POG)4 are recorded (Figure 3). The peptide mixtures BA’ and BB’ show very similar fluorescence profiles that their fluorescence after the re-equilibration at 4oC is much stronger than the original fluorescence at 4oC, suggesting that peptide B can strongly bind with A’ and B’ to form heterotrimers (Figure 3a-b). Meanwhile, the peptide mixtures BC’ and BD’ show very similar fluorescence profiles that their fluorescence after the re-equilibration at 4oC is almost the same as the original fluorescence at 4oC, suggesting that peptide B cannot form heterotrimers with C’ and D’ (Figure 3c-d). To conclude, probe peptide FAM-G(POG)8 also shows length-dependent interaction with target collagen sequences, and it displays as strong capability as FAM-G(POG)10 to bind with target collagen peptides G(POG)10 and G(POG)8.
3.5. Targeting capability of probe peptide FAM-G(POG)6 The interactions of the probe peptide FAM-G(POG)6 (denoted as peptide C) with target collagen sequences of various lengths are also investigated by comparing the fluorescence spectra of the peptide mixtures (CA’, CB’, CC’, and CD’) (Figure 4). The peptide mixtures CA’ and CB’ show a little stronger fluorescence after the re-equilibration than the original fluorescence at 4oC , suggesting a weak interaction of peptide C with A’ and B’ (Figure 4a-b). In contrast, the peptide mixtures CC’ and CD’ show very similar fluorescence after the re-equilibration at 4oC as compared to the original fluorescence at 4oC, indicating the absence of significant interactions between peptide C and peptide C’ or D’ (Figure 4c-d). Compared with FAM-G(POG)10 and FAM-G(POG)8, probe peptide FAM-G(POG)6 does not display strong interaction with target collagen peptides including G(POG)10 and G(POG)8.
3.6. Linear detection of target collage peptides by probe FAM-G(POG)8 Among the four constructed probe peptides, only FAM-G(POG)10 and FAM-G(POG)8 form homotrimers by themselves as well as heterotrimers when hybridized with target collagen sequences G(POG) 10 and G(POG)8. 8
FAM-G(POG)8 is shorter than FAM-G(POG)10, therefore it is chosen as the appropriate probe peptide to further develop a quantitative assay for target collagen sequence G(POG)10. The effect of pH and salt on the fluorescence characterization of probe peptide FAM-G(POG)8 has been first evaluated (Figure S1a). The fluorescence intensity of the probe peptide is similar at pH 7.4 and pH 8.0, while it is largely reduced at acidic pH 6.0. Compared with pH, various salt concentrations (0, 150 mM and 300 mM) result in much smaller changes in fluorescence. The fluorescence restoration capability of the target peptide G(POG) 10 is further investigated under different pH and salt conditions (Figure S1b). The recovery efficiency of G(POG) 10 on the FAM-G(POG)8 is much smaller at pH 6 than pH 7.4 and pH 8, while the presence of extra salt does not significantly affect the fluorescence. pH 7.4 PBS buffer without any salt is thus chosen as the condition for further characterization. When FAM-G(POG)8 is hybridized with various concentrations of G(POG) 10, a proportional relationship between the fluorescence of the peptide system and the concentration of the target peptide is found (R2 = 0.99) (Figure 5a). The system shows a linear range from 100 to 1000 nM, with an accurate determination of the target collagen sequence as low as 84 nM, which is comparable to the FAM-G(POG)10-based fluorescence assay with a linear range of 100-1000 nM and a detection limit of 55 nM (Figure S2a). Theses results demonstrate that the shorter peptide FAM-G(POG)8 provides a capable probe for the detection of target collagen peptide.
3.7. Detection of target collagen sequences in biologic fluids The fluorescence self-quenching assay together with the probe peptide FAM-G(POG)8 is further applied to detect target collagen sequences in human urine samples (Figure 5b). A linear relationship between the fluorescence of the peptide system and the concentration of target collagen sequence G(POG)10 is observed in the range of 100-1000 nM (R2 = 0.98) with a detection limit of 74 nM (Figure 5b). A similar proportional relationship between the fluorescence of the system and the concentration of target peptide is also identified in human saliva samples (R2 = 0.97) (Figure 5c). The FAM-G(POG)8-based fluorescence self-quenching assay has reached similar linear ranges and detection limits as the FAM-G(POG)10-based method (Figure S2b-c). The presence of target peptide G(POG)10 in human urine and saliva samples is also confirmed by Mass spectroscopy (Figure S3). These results have demonstrated that probe peptide FAM-G(POG)8 is capable to detect target collagen peptides in complex biological media. The applicability of this FAM-G(POG)8-based fluorescence self-quenching assay is evaluated in urine and saliva samples fortified with different concentrations of target peptide G(POG) 10 (Table S1). Analysis of the results of spiked samples displays the recoveries within the range of 98.7% - 108.4%, demonstrating the accuracy of the method and its suitability for routine analysis. These recoveries using the probe peptide FAM-G(POG)8 are comparable to those obtained by the assay using the probe peptide FAM-G(POG)10 (100.1% 9
- 107.9%) (Table S1). It suggests that the short peptide FAM-G(POG)8 is an excellent probe for the fluorescence self-quenching assay in the detection of target collagen peptide in biological media.
3.8. Interference The interference of other proteins in the detection of target collagen peptide sequence G(POG) 10 using probe peptide FAM-G(POG)8 is examined (Figure 6). The fluorescence intensities are measured for the probe peptide FAM-G(POG)8 in the absence and presence of nonspecific proteins BSA, hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase, respectively (purple bar). They all show relatively similar fluorescence intensities, indicating that these nonspecific proteins do not interfere with the fluorescence characterization of the probe peptide. Compared with the target peptide G(POG) 10, these proteins all exhibit much less florescence restoration capability on the FAM-G(POG)8, indicating the highly selective recognition of target collagen sequence by the probe peptide. Meanwhile, the fluorescence intensities are recorded for FAM-G(POG)8 hybridized with G(POG)10 at 518 nm in the absence and presence of nonspecific proteins (red bar). The addition of extra proteins does not significantly affect the fluorescence intensity of the hybridized peptides, indicating that the probe peptide FAM-G(POG)8 is highly specific for the target collagen sequence (Figure 6). FAM-G(POG)8 may provide a sensitive and selective probe for the detection of target collagen peptide with little interference from other proteins.
4. Conclusions As the major structural protein of human body, the degradation of collagen is involved in various diseases such as arthritis, chronic wounds and tumor [16,24]. The construction of effective assays for the detection of collagen fragments plays critical roles in diagnostic, prognostic and therapeutic decisions in collagen-related diseases. An essential question for the assay development is to discover novel 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 examined the fluorescence profiles of the probe peptides (FAM-G(POG)10, FAM-G(POG)8, FAM-G(POG)6 and FAM-G(POG)4) hybridized with various target collagen sequences, and revealed that the targeting capability of probe peptides is length-dependent. As the probe peptides become shorter, they show weaker ability to bind with the target peptides. It is observed that only FAM-G(POG)8 and peptides with higher lengths can specifically recognize target peptides. Short peptides have several advantages such as easy synthesis 10
and modification. We have further demonstrated that this newly discovered short probe peptide FAM-G(POG)8 can be applied to specifically detect the desired collagen fragment in complex biological media. The FAM-G(POG)8-based fluorescence self-quenching assay proves to be very selective for target collagen sequences with little interference from other proteins. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers. It may have very promising applications in the discovery of potent probe peptides for various protein biomarkers involved in pathological conditions.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21305056), the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2016-k10) and open fund of State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (Grant No. T151402).
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Scheme 1. Schematic Illustration of monitoring the interaction of target peptides and fluorescent probe peptides of different lengths using fluorescence self-quenching assay.
Figure 1. Fluorescence characterization of the FAM-labeled probe peptides: FAM-G(POG)10 (a), FAM-G(POG)8 (b), FAM-G(POG)6 (c), and FAM-G(POG)4 (d). Fluorescence profiles are recorded for the peptide solutions incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 2. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)10 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 3. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)8 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red). Figure 4. Fluorescence characterization of the mixture of probe peptide FAM-G(POG)6 with various target peptides G(POG)10 (a), G(POG)8 (b), G(POG)6 (c), and G(POG)4 (d). Fluorescence profiles are recorded for the peptide mixtures incubated at 4oC (black), heated to 90oC for 25 min (blue), and re-equilibrated at 4oC (red).
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Figure 5. The proportional fluorescence restoration of probe peptide FAM-G(POG)8 by the hybridization with target collagen peptide sequence G(POG)10 in PBS (a), human urine (b) and saliva (c) samples. The fluorescence emission spectra are recorded for peptide FAM-G(POG)8 in the presence of various concentrations of G(POG) 10 (0, 100, 200, 400, 600, 800,1000 nM). The fluorescence intensity monitored at 518 nm is plotted as a function of the concentration of G(POG)10. Figure 6. The interference of other proteins in the detection of target collagen peptide sequence G(POG) 10 using probe peptide FAM-G(POG)8. The fluorescence intensities are measured for probe peptide FAM-G(POG)8 at 518 nm in the absence and presence of nonspecific proteins BSA, hemoglobin, myoglobin, papin, pepsase, trypsin, bromelain and benase, respectively (purple). The fluorescence restoration of probe peptide FAM-G(POG)8 by the target peptide G(POG)10 is determined in the absence and presence of the nonspecific proteins (red).
Table 1. Design of FAM-labeled probe peptides and unlabeled target peptides with different lengths. Peptide Name
Sequence
A
FAM-G(GPO)10
B
FAM-G(GPO)8
C
FAM-G(GPO)6
D
FAM-G(GPO)4
A’
G(GPO)10
B’
G(GPO)8
C’
G(GPO)6
D’
G(GPO)4
Function
Probe
Target
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Figure 1
Figure 2
15
Figure 3
Figure 4
16
Figure 5
Figure 6
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Highlights:
The development of novel assays to detect collagen fragments is of utmost importance.
Fluorescence self-quenching assay has been established to detect collagen sequences.
The targeting ability of the probe peptides has been found to be length-dependent.
A short probe peptide has been discovered to specifically recognize target peptides.
The assay provides a powerful new tool to identify probe peptides for various biomarkers.
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