Mass spectrometry identification of age-associated proteins from the malaria mosquitoes Anopheles gambiae s.s. and Anopheles stephensi

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].


Value of the data
This study investigated the changes that occur in the head and thorax proteome of two major malaria vectors; the African and Asian malaria vectors, A. gambiae and A. stephensi respectively, during adult aging.
These changes may highlight underlying mechanisms of the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control.
A future study could use these data to identify robust age biomarkers to be incorporated into rapid age detection assays such as ELISA or dipstick assays applicable as surveillance tools in vector control settings.

Mosquito preparation and protein extraction
Two cohorts each of A. gambiae and A. stephensi were reared at different times. For each cohort, adults were collected 24 h post emergence and again at 9 and 17 d post emergence for A. gambiae and at 9, 17 and 34 d post emergence for A. stephensi. Heads and thoraces of five females at 1, 9 and 17 d of age for A. gambiae and 1, 9, 17 and 34 d for A. stephensi were dissected on a bed of dry ice and pooled into 2 ml plastic screw-cap vials containing 90 ml of 2D buffer (7 M Urea, 2 M Thiourea and 4% [w/v] CHAPS), 1 Â dissolved PhosSTOP (Roche, Penzerb, upper Bavaria, Germany) and two 3 mm silica glass beads. Proteins were extracted by homogenising samples using a Minibead-beater 96 (BioSpec Products, Inc. Bartlesville, OK, USA) for 1.45 min. The contents were collected by brief centrifugation at 12,000 Â g. The sample was transferred to new 1.5 ml micro-centrifuge tubes and clarified by centrifuging twice at 21,000 Â g for 10 min. Protein was purified using the 2D clean-up kit (GE Healthcare, Waukesha, WI, USA.) according to the manufacturer's protocol. Samples were then resuspended in 2D buffer and total protein content was quantified using the 2D quantification kit (GE Healthcare) following the manufacturer's protocol. Fifty micrograms of protein was prepared in a 2D buffer for each age sample. An internal standard sample was prepared by combining 25 mg of protein from each age sample into a single pool. The pH of all the samples was adjusted to between 8 and 9.1.
A total of four and three biological replicates of A. gambiae and A. stephensi, respectively, were included at each age and the entire experiment was replicated a second time using the second cohort of mosquitoes.

2D-DIGE
Experiments with 2D-DIGE followed the procedures described by Hugo and colleagues [2]. The Stock solutions of each of Cy2, Cy3 and Cy5 fluorescent cyanine dyes (GE Healthcare) was prepared by reconstituting the appropriate dye in 5 ml Dimethylformamide (Sigma Chemical Co. Ltd., St Louis, MO, USA.) to a concentration of 1 nM. Four hundred picomoles of either Cy3 or Cy5 was added to each sample and 2400 pmol of Cy2 was added to the internal standard. Each sample pool was combined with 4.5 ml of 100Â ampholytes (Bio-Rad, Richmond, CA, USA.), 5.6 ml Destreak reagent (GE Healthcare) and made up to 450 ml in 2D Buffer. Samples were loaded onto 24 cm, pH 3-10 immobilised pH gradient strips (Bio-Rad) in rehydration trays and left at room temperature for 4 h for the strips to absorb the sample. Following absorption, the strips were transferred to a new focussing tray, overlayed with mineral oil and allowed to rehydrate overnight at room temperature. For first dimension separation, the strips were transferred to a dry protean iso-electric focusing tray. The strips were overlayed with fresh mineral oil and iso-electric focussing was performed using the following run conditions: 50 μA per strip, 250 V for 15 min, 1000 V for 5 h, 10,000 V for 4 h and 500 V to reach a total of 80,000 V h À 1 .

Imaging and processing
A. stephensi gels were scanned using a Typhoon FLA-9400 fluorescent imager (GE Healthcare) at 100 mm pixel resolution, using the 520 band pass (BP) 40 filter for Cy2, 580 BP 30 filter for Cy3 and the 670 BP 30 filter for Cy5 emission. A. gambiae gels were scanned using a FLA-9000 Starion fluorescent imager (GE Healthcare) at 100 mm pixel resolution using the BPB1 (530DF20) filter for Cy2, DGR1 (570DF20/665LP) filter for Cy3 and LPR (665LP) filter for Cy5. Fluorescent images were processed and analysed using Delta2D version 4.0 software (Decodon, Greifswald, Germany). Spots on gel images scanned from the same gel were then connected by direct warping and the spots on all gels were aligned and normalised for intensity using the Cy2 channel (Internal standard images) on each gel with the match vectors tool. All aligned images were fused and thereafter spot validations were transferred to all other images from this fused image [3]. All spots that were visible on 2D-DIGE images for both species are shown in Supplementary tables 1-4.

Sample preparation for MS
Protein spots for identification were manually excised from 2D gels using the method of Hugo and colleagues [2]. was added to the gel plugs and incubated at room temperature, shaking for 1 h. The solution bathing the gels was combined with the previous extracts. Pooled extracts were lyophilised, rehydrated in 30 μl of 0.1% [v/v] TFA and concentrated using C18 Zip-Tips (Millipore, Billerica, MA, USA.) according to the manufacturer's instructions. In-gel tryptic digests were analysed by either MALDI-TOF/TOF MS [4] or separated by CapHPLC and sprayed directly into the ion source of LTQ-Orbitrap XL hybrid MS [5]. MS/MS spectra were automatically acquired with 40% higher laser intensity than that used for MS analysis. Spectra were calibrated using a peptide calibration standard mixture (Bruker Daltonics. Kit number 208241) of nine peptides in the mass range of m/z¼1046 and m/ z¼3147, with 62.5 fmol of each peptide applied to calibration spots. High precision MS/MS calibration was achieved using fragment ions derived from all the nine peptides in the MS calibration kit and the associated calibration coefficients were applied to the method file used to acquire MS/MS data. The data were automatically acquired and processed in a batch mode by Bruker Daltonics Flex-series software (FlexControl and FlexAnalysis version 2.4), and searched using MASCOT search engine with a custom made database using Bio-tools version 3.1. Instrument settings for MS were: ion source 1 potential, 25.00 kV; ion source 2 potential, 21.70 kV; reflectron 1 potential, 26.30 kV; reflectron 2 potential, 13.85 kV and for MS/MS were: ion source 1 potential, 8.00 kV; ion source 2 potential, 7.20 kV; reflectron 1 potential, 29.50 kV; reflectron 2 potential, 13.75 kV; LIFT 1 voltage, 19.00 kV; LIFT 2 voltage, 3.00 kV. Ion selector resolution was set at 0.5% of the mass of the precursor ion [6]. All protein spots identified by MALDI-TOF/TOF mass spectrometry are reported in our accompanying manuscript [1].

Liquid chromatography (LC)-MS/MS analysis
In-gel tryptic digests were fractionated by CapHPLC using a Shimadzu Prominence HPLC system (Shimadzu) and were introduced directly into the LTQ-Orbitrap XL hybrid MS (Thermo Fisher Scientific, Bremen, Germany) equipped with a dynamic nanoelectrospray ion source (Proxeon, Odense, Denmark) and distal coated silica emitters (30 μm i.d., 20 μm tip i.d; New Objective, Woburn, MA, USA.). Acidified samples were loaded onto a 120 Å, 3 μm particle size, 300 μm by10 mm C18-AQ Reprosil-Pur trap column (SGE Australia Pty., Ltd.) at 30 μl/min in 98% solvent A (0.1% [v/v] aqueous formic acid) and 2% solvent B (80% [v/v] acetonitrile containing 0.1% [v/v] formic acid) for 3 min at 40 1C, and were subsequently gradient eluted onto a pre-equilibrated self-packed analytical column (Dr. Maisch GmbH Reprosil-Pur C18-AQ, 120 Å, 150 μm by 200 mm) using a flow rate of 900 nl/min. The LTQ-Orbitrap was controlled using Xcalibur 2.0 SR1 (Thermo Fisher Scientific). Analyses were carried out in data-dependent acquisition mode, whereby the survey full scan mass spectra (m/z 300-2000) were acquired in the Orbitrap FT mass analyser at a resolution of 60,000 (at 400m/z) after accumulating ions to an automatic gain control target value of 5.0 Â 10 5 charges in the LTQ mass analyser. MS/MS mass spectra were concurrently acquired on the eight most intense ions in the full scan mass spectra in the LTQ mass analyser to an automatic gain control target value of 3.0 Â 10 4 charges. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion (repeat count, 1; repeat duration, 30 s; exclusion list size, 500; and exclusion duration, 90 s) were used. Fragmentation conditions in the LTQ were: 35% normalised collision energy, activation q of 0.25, isolation width of 3.0 Da, 30 ms activation time, and minimum ion selection intensity 500 counts. Maximum ion injection times were 500 ms for survey full scans and 100 ms for MS/MS [3,[5][6][7]. All spots identified by LC-MS/MS are shown in Fig. 1 and their identities are presented in Table 1.  A mass tolerance of 20 ppm for peptide precursor ions was applied in all searches. Multiple protein hits were typically achieved from spot identifications using CapHPLC; possibly due to inefficient isoelectric focusing or electrophoresis and/or high sensitivity of the LTQ-Orbitrap. Therefore, only protein hits with estimated size and charge states within arbitrary ranges of 75 kDa and 7 1 pI from the actual spot positions were included in the table. At least two peptide identifications were required to assign protein identification. a A. stephensi proteins pots as shown in Fig. 1A. b A. gambiae protein spot as shown in Fig. 1B. 22/05/2012 and from VectorBase database on 26/05/14. A mass tolerance of 100 ppm for peptide precursor ions and 0.8 Da for fragment ions was applied in all searches except for Orbitrap dataset where a precursor ion tolerance of 20 ppm was applied. Database search parameters for both MALDI-TOF/TOF and LTQ-Orbitrap datasets were as follows: enzymatic cleavage, tryptic; fixed modifications, S-carboxamidomethylation of cysteine residues; variable modifications, methionine oxidation, deamidation of asparagine and glutamine; and missed cleavages. Peptides identified by TOF/TOF mass spectrometry were reported as significant scoring peptides at a MASCOT probability based score threshold of p o0.05 (o5% chance to be a false positive hit). For the Orbitrap datasets, to estimate the false discovery rate (FDR) at which the identification was considered correct (q-Value), a Percolator algorithm implemented in Proteome Discoverer (version 1.4.0.288, Thermo Fisher Scientific) was used and proteins with peptides having a confidence threshold q-Value o0.01 (1% FDR) were considered to be valid. A minimum of two confident peptide identifications were required to assign a protein identity. Putative function of hypothetical proteins was inferred using the VectorBase database.