Proteolysis in Mixed Organic−Aqueous Solvent Systems:  Applications for Peptide Mass Mapping Using Mass Spectrometry

Russell, William K.; Park, Chul–Seung; Russell, David H.

doi:10.1021/ac001332p

Cited by 268 publications

(281 citation statements)

References 38 publications

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“…Although the digestion efficiency of this bioreactor was high, not all of the peptides were identified, which may be due to incomplete digestion for protease-inaccessible proteins, solubility of digested peptides, and lack of a denaturation step in the bioreactor protocol [39]. Sequence coverage ranging from 18% to 95% have been reported for in-solution digestion of cytochrome c, which requires 15 min to 24 h reaction time [25,29,40].…”

Section: Maldi Analysis Of Solid-phase Bioreactor Digested Cytochrome Cmentioning

confidence: 99%

“…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-…”

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confidence: 99%

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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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Section: Maldi Analysis Of Solid-phase Bioreactor Digested Cytochrome Cmentioning

confidence: 99%

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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“…For IZ stain, destain was performed by 2 Â 8 min incubation in 1 mL 50 mM Tris buffer, 0.3 M glycine, pH 8.3 containing 30% ACN. While, for EY stain, the gel pieces were destained with 30% EtOH for 1 h. In-gel digestion was performed in three steps following the protocol modified by Russell et al [27]. Briefly, the gel slices were washed with DW and 50% ACN and incubated with 100% ACN for 10 min.…”

Section: Maldi-tof Msmentioning

confidence: 99%

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

et al. 2010

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A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, lowcost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.

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“…A 50 L aliquot was removed and mixed with 50 L of ACN to improve proteolysis [23]. Trypsin was added to a final protease-to-BSA ratio of 1:40, and the digestion mixture was incubated at 37°C for 24 h. The resulting digested BSA sample was used to prepare 10-, 100-, and 1000-fold serial dilutions in water (i.e., protein concentrations of 100, 10, and 1 ng/ L, respectively).…”

Section: Digestion Of Bsamentioning

confidence: 99%

Factors that affect ion trap data-dependent MS/MS in proteomics

Wenner

Lynn

2004

J. Am. Soc. Mass Spectrom.

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Quadrupole ion trap scanning parameters for performing bottom-up proteomics in a datadependent fashion were evaluated on a Finnigan LCQ Deca mass spectrometer. Evaluation of parameters such as the number of averaged full scans, the number of averaged MS/MS scans, and ion injection times were necessary for acquiring high quality MS/MS spectra that yield favorable b and y ion coverage and high correlation to proteins using database searching algorithms. In this study, we demonstrated how the duty cycle of the mass spectrometer affects the number of peptides that can be successfully identified by SEQUEST using a model system of tryptic BSA peptides to mimic a typical complex mixture associated with bottom-up proteomics. The number of averaged scans and the duration of ion accumulation in the trap had a significant effect on the quality of acquired MS/MS spectra. For example, by increasing the ion injection time from 500 ms to 600 ms, peptide HLVDEPQNLIK improved from being improperly identified to being correctly identified with a SEQUEST cross-correlation score of 3.60. As a result of these experiments, we have devised the following set of ion trap parameters for performing bottom-up proteomics analysis in our laboratory: Three averaged full scans, five averaged MS/MS scans, and a maximum ion injection time of 600 ms. (J Am Soc Mass Spectrom 2004, 15, 150Ϫ157)

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Proteolysis in Mixed Organic−Aqueous Solvent Systems: Applications for Peptide Mass Mapping Using Mass Spectrometry

Cited by 268 publications

References 38 publications

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

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

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

Factors that affect ion trap data-dependent MS/MS in proteomics

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