2013
DOI: 10.1021/pr400835k
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Quantitative Phosphoproteomics Using Acetone-Based Peptide Labeling: Method Evaluation and Application to a Cardiac Ischemia/Reperfusion Model

Abstract: Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. In this report, an MS-based strategy utilizing an inexpensive acetone-based peptide labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Since the chemistry for RABA-labeling for phosphorylation profiling had not… Show more

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Cited by 13 publications
(13 citation statements)
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“…The enriched phosphopeptides were eluted with 50 µL of elution buffer (30% ammonium hydroxide solution in water) twice. The eluted enriched phosphopeptides were dried using speed vac and stored at −80 °C before further analysis as described previously 9 .…”
Section: Methodsmentioning
confidence: 99%
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“…The enriched phosphopeptides were eluted with 50 µL of elution buffer (30% ammonium hydroxide solution in water) twice. The eluted enriched phosphopeptides were dried using speed vac and stored at −80 °C before further analysis as described previously 9 .…”
Section: Methodsmentioning
confidence: 99%
“…For the analytical/chromatographic separation of phosphopeptides, the trap column was switched to align with the nanoLC separation column, an Acclaim PepMap 100 (inner diameter 75 µm, length 15 cm, C18 particle size of 3 µm, 100 A of pore size) from Dionex-Thermo Fisher Scientific (Sunnyvale, CA). The peptide elution step was achieved using a varying mobile phase gradient from 95% phase A (0.1% formic acid in water, v/v) to 40% phase B (0.1% formic acid in acetonitrile, v/v) for 70 min, from 40% phase B to 85% phase B for 5 min, next keeping the same mobile phase composition for 5 min at 300 nL/min as described previously 9 .…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…Nano-LC-MS/MS analyses were performed on a TripleTOF 56001 (SCIEX, Toronto, Ontario, Canada) attached to an Eksigent (Dublin, Calif) nano-LC/ultra-nanoflow system, as described previously, 42 but with the following changes: (1) LC mobile phases included 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B; (2) samples were trapped and desalted on an Eksigent NanoLC Trap (ChromXP C18-CL-3 mm 120 A, 350 mm 3 0.5 mm) at 1 mL/min in 0/1% formic acid for 15 minutes; and (3) all data were recorded with Analyst TF software (version 1.7, build 96).…”
Section: Dapa and Mass Spectrometrymentioning
confidence: 99%
“…A variety of enrichment and fractionation methods in phosphoproteomics have been reported including the enrichment with immobilized metal affinity chromatography (IMAC) 6,[14][15][16] and metal oxide affinity chromatography (MOAC) as well as titanium dioxide (TiO 2 ), [17][18][19][20][21][22][23][24][25] and fractionation with strong cation exchange (SCX), 26,27 hydrophilic interaction chromatography (HILIC), [28][29][30] and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). [31][32][33] More recently, the two sequential steps of preparation methods are commonly used in large scale phosphoproteomics.…”
Section: Introductionmentioning
confidence: 99%