An integrated high-performance liquid chromatography–mass spectrometry system for the activity-dependent analysis of matrix metalloproteases (original) (raw)
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Journal of Analytical Sciences, Methods and Instrumentation, 2013
Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose but immunoassays face limitation of both availability and qualities of antibody reagents that are necessary for development of immune assays. With genomics data base available, mass spectrometry (MS) can analyze protein tryptic peptides directly for quantitative determination of proteins. In this study, we report a method for detection of matrix metalloproteinase 1 (MMP1), an important extracellular matrix modulator, in human breast cancer cells by quadrupole time-of-flight (Q-TOF) MS. Absolute quantification of MMP1 was conducted using the selected reaction monitoring (SRM) on a triple quadrupole (Triple-Quad) MS via transitions selected from MMP1 tryptic peptides using non isotope labeled MMP1 protein as a titration standard. In comparison with immune based assay, this MS method showed picogram level sensitivity for quantitative determination of MMP1 in total cell lysates. Our results demonstrated the feasibility of absolute quantification of low abundance proteins using label-free protein standard by mass spectrometry. Therefore, this method provides not only advantages of high sensitivity but also cost saving in comparison with the commonly used mass spectrometry that currently employs isotype labeled proteins for quantitative analysis.
Chemistry & Biology, 2006
Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, are implicated in tumor progression. We describe herein the development of a resin-immobilized, broad-spectrum synthetic MMP inhibitor for selective binding of the active forms of MMPs from different experimental samples. We confirmed the activity-based binding of MMPs to the inhibitor-tethered resin with purified human recombinant MMP-2, -9, and -14, samples of cultured cells, and tissue extracts. Our results show that only the free active MMPs, and not the zymogens or MMP/TIMP (enzyme-protein inhibitor) complexes, bound specifically to the resin. In our comparison of benign and carcinoma tissue extracts, we detected active MMP-2 and MMP-14 forms only in the cancerous tissue samples, indicating that a pool of the tumor MMPs is free of endogenous inhibitors (TIMPs), and is thus likely to contribute to proteolytic events that precipitate tumor metastasis.
As principal degrading enzymes of the extracellular matrix, metalloproteinases contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern metalloproteinase activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (<20μl) of complex biological fluids such as clinical samples which are only available in very limited amounts. We have developed a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library containing mixtures of unique moderately selective FRET-based protease substrates and specific inhibitors and monitors hundreds of the reactions thus initiated simultaneously by tracking these droplets. Specific protease activities in the sample are then inferred from the reaction rates using a deconvolution technique, Proteolytic Activity Matrix Analysis (PrAMA). Using a nine-member droplet library with three inhibitors and four FRET substrates, we apply the method to the peritoneal fluid of subjects with and without the invasive disease of endometriosis. Results show clear and physiologically relevant differences with disease; in particular, decreased MMP-2 and ADAM-9 activities. Extracellular proteases participate in myriad physiological and disease processes, most prominently by degrading extracellular matrix components. In particular, matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) have been investigated as potential drug targets and diagnostic biomarkers. Metalloproteinase activities are regulated through a tight network of multiple proteolytic enzymes and inhibitors
Molecular Biotechnology, 2014
Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed 'form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol. have contributed equally to this work.
Matrix biology : journal of the International Society for Matrix Biology, 2015
Matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Site (PICS) method to simultaneously profile both the prime and nonprime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with an ~32-93% preference for leucine in P1' depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptid...
Comparison of metalloproteinase protein and activity profiling
Analytical Biochemistry, 2011
Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.
Molecular & Cellular Proteomics, 2006
The advances in proteomic technologies provide tremendous opportunities for applying these technologies in biomarker-related clinical applications; however, the unique characteristics of human biofluids such as high dynamic range in protein abundances and extreme complexity of human proteomes present tremendous challenges for current analytical technologies. In this review, we focus on summarizing the recent advances in LC-MS based proteomic profiling and
Frontiers in Immunology
The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA ® technology. We describe two procedures: (i) one approach is used to analyze MMP-9-TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9-TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9-TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9-TIMP-1 AlphaLISA ® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9-TIMP-1 complexes in circulating blood as biofluid.
The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics
Methods and Protocols
The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment in...
bioRxiv (Cold Spring Harbor Laboratory), 2024
Multi-step multi-hour tryptic proteolysis has limited the utility of bottom-up proteomics for cases that require immediate quantitative information. The recently available hyperthermoacidic (HTA) protease "Krakatoa" digests samples in a single 5 to 30-minute step at pH 3 and >80 C; conditions that disrupt most cells and tissues, denature proteins, and block disulfide reformation. The combination of quick single-step sample preparation with high throughput dual trapping column single analytical column (DTSC) liquid chromatography-mass spectrometry (LC-MS) achieves "Rapid Proteomics" in which the time from sample collection to actionable data is less than 1 hour. The presented development and systematic evaluation of this methodology found reproducible quantitation of over 160 proteins from just 1 microliter of whole blood. Furthermore, the preference of the HTA-protease for intact proteins over peptides allows for sensitive targeted quantitation of the Angiotensin I and II bioactive peptides in under half an hour. With these methods we analyzed serum and plasma from 53 individuals and quantified Angiotensin and proteins that were not detected with trypsin. This assessment of Rapid Proteomics suggests that concentration of circulating protein and peptide biomarkers could be measured in almost real-time by LC-MS.