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RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected. ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.
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Ultra‐low current electrospray ionization of chloroform solution for the analysis of perfluorinated sulfonic acids
RationaleFemtoamp and picoamp electrospray ionization (ESI) characteristics of a nonpolar solvent were explored. The direct ESI mass spectrometry analysis of chloroform extract solution enabled rapid analysis of perfluorinated sulfonic acid analytes in drinking water. MethodsNeat chloroform solvent and extracts were directly used in a typical wire‐in ESI setup using micrometer emitter tips. Ionization currents were measured with femtoamp sensitivity while ramping the spray voltage from 0 to −5000 V. Methanol was used as a comparison to illustrate the characteristics of electrospraying chloroform. The effects of spray voltage and inlet temperature were studied. A liquid–liquid extraction workflow was developed to analyze perfluorooctanoate sulfonate (PFOS) in drinking water using an ion‐trap mass spectrometer. ResultsThe ionization onset of chloroform solution was 41 ± 17 fA at 300 V. The ionization current gradually increased with voltage while remaining below 100 pA when using voltages up to −5000 V. The ion signal of PFOS was significantly enhanced to improve the limit of detection (LoD) to 25 ppt in chloroform. Coupled with a liquid–liquid extraction workflow, LoD of 0.38–5.1 ppt and a quantitation range of 5–400 ppt were achieved for perfluorinated sulfonic compounds in 1‐ml water samples. ConclusionsFemtoamp and picoamp modes expand the solvent compatibility range of ESI and can enable quantitative analysis in parts per trillion (ppt) concentrations.
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- Award ID(s):
- 2203289
- PAR ID:
- 10403153
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Rapid Communications in Mass Spectrometry
- Volume:
- 37
- Issue:
- S1
- ISSN:
- 0951-4198
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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