A triaxial probe for on-line proteolysis coupled with hydrogen/deuterium exchange-electrospray mass spectrometry

Chen, Maolian; Cook, Kelsey D.; Kheterpal, Indu; Wetzel, Ronald

doi:10.1016/j.jasms.2006.09.011

Cited by 15 publications

(24 citation statements)

References 38 publications

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“…The standard Q-Star IonSpray™ probe can be used in both a triaxial and a coaxial mode; details of these modes are provided elsewhere [19]. Diameters and flows for the various tubes were as indicated in Table 1.…”

Section: Electrospray Mass Spectrometrymentioning

confidence: 99%

“…Indicated uncertainties in relative intensities (or derived quantities) represent the standard deviation of 3 replicate spectra, each of which comprised 60 scans. To avoid interference from atmospheric moisture in hydrogen/deuterium exchange studies [19], the hole from the source leading to the venturi pump is generally blocked with a rubber stopper and nitrogen gas (500 mL/min, from boil-off) introduced into the source through the "make-up" hole in the source housing during and for 4 hours before sample injection. Although oxidation did not appear to be affected, this precaution was practiced here for consistency with earlier studies.…”

Section: Electrospray Mass Spectrometrymentioning

confidence: 99%

“…This "heavy" peak was also observed for other charge states (+4: 78% ± 6%; +6: 82% ± 4%) ( Figure 1a). On-line pepsin hydrolysis (as described in [19]) was undertaken to help identify the "heavy" peak. The peaks attributable to oxidized species were much less intense in the coaxial mode (compare Figures 1b and 1c, both obtained with emitter A), and the emitter current was much lower (~2.2 µA) and more stable.…”

Section: Identification Of Peptide Oxidationmentioning

confidence: 99%

“…In light of the general difficulty of spraying pure aqueous solutions with conventional (as opposed to micro-or nanospray) emitters [26], one possible contributor to the excess oxidation in the triaxial mode is the imperfect mixing that is achieved in that mode [19]. To assess this contribution, Aβ samples were premixed to the same final composition as that used in the coaxial probe (50.5/49.5/0.46 water/MeCN/formic acid), then pumped through both Tubes 1 and 2 with the probe in the triaxial configuration.…”

Section: Differences Between the Triaxial And Coaxial Modesmentioning

confidence: 99%

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Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

2007

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Gradual corrosion of stainless steel electrospray emitters under conditions of normal use generates surface irregularities that can promote electrical discharge. The increased emission current affects the electrochemical reactions associated with the spray process. When sampling the peptide Aβ(1-40), this is manifest by oxidation of methionine at position 35 to methionine sulfoxide. The resultant mass shift and reduced sensitivity can adversely affect H/D exchange experiments. These effects can be avoided by adding a redox buffer or (preferably) by re-polishing the emitter, especially to a rounded geometry.

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Section: Electrospray Mass Spectrometrymentioning

confidence: 99%

Section: Electrospray Mass Spectrometrymentioning

confidence: 99%

Section: Identification Of Peptide Oxidationmentioning

confidence: 99%

Section: Differences Between the Triaxial And Coaxial Modesmentioning

confidence: 99%

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Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

2007

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“…However, the long sample incubation times required for trypsin digestion in solution and the extensive sample treatment steps result in long protein processing times. To overcome those obstacles, rapid in-solution digestion protocols have been developed using organic solvents to denature proteins or by applying higher incubation temperature to accelerate reaction time [27][28][29][30]. However, efficient protein identifi-…”

mentioning

confidence: 99%

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Lee

Musyimi

Soper

et al. 2008

J. Am. Soc. Mass Spectrom.

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An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 ILm wide, 100 ILm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethy1carbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 ILL/min, producing a reaction time of -24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, Significant progress has been made toward the development of microchip-based technologies for proteomics through the integration of analytical processes into platforms that can provide rapid identification of proteins and the subsequent characterization of various post-translational modifications [1][2][3][4]. The small sample and reagent requirements, rapid analysis times, high throughput processing capabilities, and low operating costs are among the driving forces for the development of these systems [5][6][7][8]. Different microfluidic devices have been applied to specific aspects of protein processing, in particular, protein purification and separation, protein digestion, and protein identification by mass spectrometry [9].There have been a number of approaches to on-line and off-line matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) integrated to chipbased devices [10][11][12][13][14][15][16][17][18]. The primary advantage of the MALDI approach compared with electrospray ionization (ESI) when coupling to microfluidic chips is the potential for multiplexing [19]. The directional control Address reprint requests to Dr. Kermit K. Murray, Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70802, USA. E-mail: kkmurray@!su.edu of the ESI spray can be difficult with a high-density array of spray tips. Furthermore, microfluidic chip interfaces to ESI can suffer from stability problems when sprays are started or stopped [20,21]. This limits the speed of moving from one sample to another and therefore, limits throughput.With recent advances in analytical methods for proteomics, attention has been directed toward development of effic...

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Gaining Insight Into Reactivity Differences Between Malonic Acid Half Thioesters (MAHT) and Malonic Acid Half Oxyesters (MAHO)

Bew

Stephenson

Rouden

et al. 2017

Chemistry A European J

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An efficient two-step synthesis of structurally and functionally diverse thiophenol- and (cyclo)alkyl-derived malonic acid half thioesters (MAHTs) and phenol-derived malonic acid half oxyesters (MAHOs) has been achieved using cheap, readily available and easily handled starting materials. The synthesis of the MAHTs and MAHOs (the majority of which have not been previously reported) is readily scalable to afford gram quantities of product. In a hydrogen→deuterium exchange, an interesting stereoelectronic effect was observed when different aryl groups were incorporated. Significant changes in the rates of hydrogen→deuterium exchange and levels of isotope incorporation were observed. By way of example, using [ H]methanol and 4-bromophenol-derived MAHO afforded only 14 % [ H]-incorporation (9 min, k=31) whereas the corresponding 4-bromothiophenol-derived MAHT afforded 97 % [ H]-incorporation (9 min, k=208). In a benchmark procedure and comprehensive DFT study, 54 ester and thioester configurations and conformations were characterized. In the MAHT series, a sulfur-containing molecular orbital provides a path for increased delocalisation of electron density into the enol that is unavailable in MAHOs. This facilitates keto-enol tautomerisation and consequently enhances the rate and percentage of hydrogen→deuterium exchange.

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A triaxial probe for on-line proteolysis coupled with hydrogen/deuterium exchange-electrospray mass spectrometry

Cited by 15 publications

References 38 publications

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Gaining Insight Into Reactivity Differences Between Malonic Acid Half Thioesters (MAHT) and Malonic Acid Half Oxyesters (MAHO)

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