Capillary LC-FT-MS/MS Proteomic Analysis

Capillary LC-FT-MS/MS was performed as described by Raphael et al. (2014) [24 (link)] using a splitless nanoLC-2D pump (Eksigent, CA), a 50 µm-i.d. column packed with 7 cm of 3 µm-o.d. C18 particles, and a hybrid linear ion trap-Fourier-transform tandem mass spectrometer (LTQ-ELITE; Thermo Fisher, CA). The reverse-phase gradient was 2 to 62% of 0.1% formic acid (FA) in acetonitrile over 60 min at 350 nL/min. The top six most abundant eluting ions were fragmented by data-dependent high-energy collision-induced dissociation (HCD). Probability-based and error-tolerant protein database searching of MS/MS spectra against the Trembl protein database (release of 2013) were performed with a 10-node Mascot cluster (version 2.3.02, Matrix Science, London, UK). Search criteria included peak picking with Mascot Distiller, 10 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, 3 missed cleavages, trypsin, carbamidomethyl cysteines and oxidized methionines as variable modifications, an ion score threshold of 20, and TMT-6-plex for quantification.

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Ortiz C., Morales L., Sastre M., Haskins W.E, & Matta J. (2016). Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A. Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 3696232.

Publication 2016

splitless nanoLC-2D pump Mascot cluster

Acetonitrile Capillary Cleavages Cysteines Formic acid Hybrid Methionines Ms ms Tolerance Trypsin

Corresponding Organization : Ponce Health Sciences University

Other organizations : University of Puerto Rico at Humacao, The University of Texas at San Antonio

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Variable analysis

independent variables

Reverse-phase gradient (2 to 62% of 0.1% formic acid in acetonitrile over 60 min)
Flow rate (350 nL/min)

dependent variables

Eluting ions fragmented by data-dependent high-energy collision-induced dissociation (HCD)
Protein identification and quantification from MS/MS spectra

control variables

50 µm-i.d. column packed with 7 cm of 3 µm-o.d. C18 particles
Hybrid linear ion trap-Fourier-transform tandem mass spectrometer (LTQ-ELITE)
Probability-based and error-tolerant protein database searching against Trembl protein database (release of 2013)
Search criteria: peak picking with Mascot Distiller, 10 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, 3 missed cleavages, trypsin, carbamidomethyl cysteines and oxidized methionines as variable modifications, ion score threshold of 20, and TMT-6-plex for quantification

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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