Pooled hemolymph from 400–500 naïve or challenged mosquitoes were loaded onto a hydrophilic interaction column (PolyHYDROXYETHYL A, 100 × 2.1 mm ID, 300-Å pore, PolyLC Inc., Columbia, MD) equilibrated with 90% of acetonitrile (LC/MS quality acetonitrile, acid free, Fisher Scientific) using the AKTA purifier system (GE Healthcare, Piscataway, NJ). Fractions were eluted with a linear gradient of 90-0% acetonitrile in water over 30 min at a flow rate of 0.25 ml/min. Absorption of the eluate was monitored at 225 and 280 nm. Bioactive fractions were dried under vacuum and sent for LC/MS analysis using an Agilent 1100 nanoflow LC system (Agilent Technologies, Palo Alto, CA) coupled online with a linear ion-trap (LIT) mass spectrometer (LTQ, ThermoElectron, San José, CA). Nanoflow reverse-phase liquid chromatography coupled with tandem MS (MS/MS) was performed as described before27 (link). Tandem mass spectra were searched using SEQUEST on a 20-node Beowulf cluster against the NR NCBI proteome database with methionine oxidation included as dynamic modification.
Agilent 1100 nanoflow lc system
Manufactured by Agilent Technologies
Sourced in Canada
The Agilent 1100 nanoflow LC system is a liquid chromatography instrument designed for high-performance nanoflow separations. It features a nanoflow pump, autosampler, and other components necessary for the analysis of small sample volumes at low flow rates.
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3 protocols using agilent 1100 nanoflow lc system
1
Mosquito Hemolymph Proteomic Analysis
2
Mosquito Hemolymph Proteomic Analysis
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Pooled haemolymph from 400–500 Naive or Challenged mosquitoes were loaded onto a hydrophilic interaction column (PolyHYDROXYETHYL A, 100 × 2.1 mm ID, 300-Å pore, PolyLC Inc., Columbia, MD) equilibrated with 90% of acetonitrile (LC/MS quality acetonitrile, acid free, Fisher Scientific) using the AKTA purifier system (GE Healthcare, Piscataway, NJ). Fractions were eluted with a linear gradient of 90-0% acetonitrile in water over 30 min at a flow rate of 0.25 ml min−1. Absorption of the eluate was monitored at 225 and 280 nm. Bioactive fractions were dried under vacuum and sent for LC/MS analysis using an Agilent 1,100 nanoflow LC system (Agilent Technologies, Palo Alto, CA) coupled online with a linear ion-trap mass spectrometer (LTQ, ThermoElectron, San José, CA). Nanoflow reverse-phase LC-MS/MS was performed as described before28 (link). Tandem mass spectra were searched using SEQUEST on a 20-node Beowulf cluster against the NR NCBI proteome database with methionine oxidation included as dynamic modification.
3
Proteomics Sample Preparation and LC-MS/MS Analysis
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TCA precipitated and acetone washed protein pellets were dissolved in 0.5 M Tris–HCl pH 8.6, 6 M guanidinium hydrochloride, reduced in 16 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 min, and alkylated in 35 mM iodoacetamide for 30 min in the dark. The proteins were digested at 37°C with trypsin (Promega, Madison, USA) after 6× dilution in 50 mM Tris–HCl pH 7.4, 5 mM CaCl2 overnight. The resulting peptides were separated on a 75 μm × 10 cm Magic C18 column (Michrom, Bioresources, Auburn, USA) with an Agilent 1100 Nanoflow LC System (Agilent, Palo Alto, CA, USA). The LC was connected to a LTQ Orbitrap Velos (Thermo Scientific). Mascot (Matrix Science, London, UK) searching UniProt data base version 2012_09 was used to identify the peptides.
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