A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue

Jiang, Hao; Yuan, Huan; Liang, Yu; Xia, Simin; Zhao, Qun; Wu, Qi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

doi:10.1016/j.chroma.2012.03.014

Cited by 22 publications

(10 citation statements)

References 33 publications

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“…2.0–2.4 µg [59], [60]); Sun et al reported ∼220 protein IDs when working with similar protein amounts, employing off-line immobilized trypsin digestion [61]. Other central Wnt-pathway proteins were readily identified in the comprehensive mode; such as β-catenin, low density lipoprotein receptor-related protein 6 (LRP5/6), disheveled segment polarity protein 1 (DVL), and casein kinase 2 (CK2), showing that untargeted OTER-PLOT can provide useful complementary information within reasonable time.…”

Section: Resultsmentioning

confidence: 99%

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

et al. 2014

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A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.

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Section: Resultsmentioning

confidence: 99%

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

et al. 2014

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“…To determine the amount of immobilized trypsin on the monolith, NaOH solution was used to cleave trypsin from the support [11,23,36], and then the supernatant was analyzed by BCA assay [5]. In our experiment, it was calculated that 1.44 ± 0.01 g (n = 3) of trypsin was immobilized on a 1 cm length microreactor, as shown in Fig.…”

Section: Characterization Of Maa-silica Hybrid Monolith Based Imermentioning

confidence: 99%

“…Furthermore, the enzymatic activity of such an IMER was determined by the hydrolysis of BAEE. The collected products were analyzed spetrophotometrically, and more than 1500 BAEE-U could be obtained, comparable to that obtained by a hydrophilic NVP modified polymer particle based IMER with the same dimension [5].…”

Section: Characterization Of Maa-silica Hybrid Monolith Based Imermentioning

confidence: 99%

“…The supernatant was collected as the soluble fraction of the extracted E. coli and MHCC97L whole cell lysate proteins. Finally, the protein concentration was determined by BCA assay, and the proteins were reduced by DTT, alkylated by IAA and desalted by a C8 precolumn as mentioned elsewhere [5]. The obtained protein fractions were lyophilized by a Speed Vac Concentrator (Thermo, San Jose, CA), and the pellets were stored at −20 • C before use.…”

Section: Sample Preparationmentioning

confidence: 99%

“…The enzyme activity of the IMER was determined according to our previous protocol [5]. Briefly, 20 mM N ␣ -benzoyl-l-arginine ethyl ester hydrochloride (BAEE) in 20 mM NH 4 HCO 3 (pH 8.0) was used as a substrate, and introduced into a 2 cm-length IMER at the flow rate of 1 L/min.…”

Section: Enzyme Activity Assaymentioning

confidence: 99%

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Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid–silica hybrid monolith as matrix for on-line protein digestion

Yuan

Zhang

2014

Journal of Chromatography A

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An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling

Weng¹,

Qu²,

Jiang³

et al. 2014

Analytica Chimica Acta

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A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue

Cited by 22 publications

References 33 publications

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid–silica hybrid monolith as matrix for on-line protein digestion

An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling

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