This work designed a mass spectrometric biosensing strategy for the multiplex detection of matrix metalloproteinases (MMPs) with a mass-encoded suspension array. This array was fabricated as multiplex sensing probes by functionalizing magnetic beads with MMP-specific peptide-isobaric tags for relative and absolute quantification (iTRAQ) conjugates, which contained a hexahistidine tag for surface binding, a substrate region for MMP cleavage, and a coding region for the specific MMP. The integration of the multiplex coding ability of iTRAQ with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the proteolysis method for peptide digestion endowed the biosensing method with high throughput and ultrahigh sensitivity. This strategy could be conveniently performed by mixing the sample and the suspension array for enzymatic reactions and then digesting the uncleaved peptides with trypsin to release the coding regions for UPLC-MS/MS analysis. With MMP-2 and MMP-7 as analytes, the relative changes of peak area ratios of coding regions showed good linear responses in the ranges of 0.2− 100 and 0.5−400 ng mL −1 , with detection limits of 0.064 and 0.17 ng mL −1 , respectively. The analysis of MMP activity in serum samples and its change responding to inhibitors demonstrated the specificity, practicability, and expansibility of the proposed strategy. This work paves a new avenue for the activity assays of multiplex enzymes and promotes the development of mass spectrometric biosensing.

Our findings also lay a theoretical foundation for the control, diagnosis, and prognosis of pathogenic E. coli related diseases.

Objective: To clone human IL-16 gene, and construct its prokaryotic expression vector, and express it in E. coli DH5. Methods: The total RNA was extracted from peripheral blood mononuclear cells stimulated by histamine. And hIL-16 cDNA was amplified by using RT-PCR and cloned into pUC18 T-vector. After the sequence of hIL-16 cDNA was confirmed, the cDNA was inserted into prokaryotic expression vector pMAL-C2. The recombinant expression plasmid pMAL-IL-16 was identified by endonucleases digestion and PCR, and then transformed into E. coli DH5, human IL-16 expressed in DH5 was identified by SDS-PAGE and Western blotting method. Results: Obtained cDNA of hIL-16 was identical with that published on Genebank, and the prokaryotic expression vector pMAL-IL-16 was constructed correctly. Recombinant protein was expressed in E.coli when induced with IPTG and had a molecular weight of 60kD, wich is consistent with theoretical calculation. Conclusion: The hIL-16 cDNA was cloned and the prokaryotic expression plasmid pMAL-IL-16 was constructed successfully. The E. coli DH5 that stably expressed hIL-16 was obtained, which provide experimental basis for the further studying of functional activities and application of hIL-16. [Key words] Human interleukin-16, Molecular cloning, Prokaryotic expression

The detection of matrix metalloproteinases (MMPs) is of great importance for diagnosis and staging of cancer. This work proposed a signal-on mass spectrometric biosensing strategy with a phospholipid-structured mass-encoded microplate for assessment of multiplex MMP activities. The designed substrate and internal standard peptides were subsequently labeled with the reagents of isobaric tags for relative and absolute quantification (iTRAQ), and DSPE-PEG(2000)maleimide was embedded on the surface of a 96-well glass bottom plate to fabricate the phospholipid-structured mass-encoded microplate, which offered a simulated environment of the extracellular space for enzyme reactions between MMPs and the substrates. The strategy achieved multiplex MMP activity assays by dropping the sample in the well for enzyme cleavages, followed by adding trypsin to release the coding regions for ultrahigh performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) analysis. The peak area ratios of released coding regions and their respective internal standard (IS) peptides exhibited satisfied linear ranges of 0.05–50, 0.1–250, and 0.1–100 ng mL–1 with the detection limits of 0.017, 0.046, and 0.032 ng mL–1 for MMP-2, MMP-7, and MMP-3, respectively. The proposed strategy demonstrated good practicability in inhibition analysis and detections of multiplex MMP activities in serum samples. It is of great potential for clinical applications and can be expanded for multiplex enzyme assays.