Thoraxes, wings and legs were manually homogenized in 10 μL of 70% formic acid and 10 μL of 50% acetonitrile (Fluka, Buchs, Switzerland) in 1.5 ml microtubes using pellet pestles (Fischer Scientific, Strasbourg, France).The homogenates were centrifuged at 10,000 rpm for 20 seconds, and 1 μL of the supernatant of each sample, corresponding to protein extract, was deposited onto a steel target plate (Bruker Daltonics, Wissembourg, France) in four spots for each sample as previously described [40 (link)]. Subsequently, 1 μL of matrix composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile(v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water was directly overlaid on each spot sample on the target plate, dried for several minutes at room temperature and placed in the MALDI-TOF MS instrument for analysis (S1 File ). To control loading on mass spectrum steel, matrix quality and MALDI-TOF apparatus performance, the matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I).
Protein calibration standard 1
Manufactured by Bruker
Sourced in Germany, France, United States
The Protein Calibration Standard I is a laboratory equipment product designed to provide a set of well-characterized proteins for calibrating and validating protein analysis instruments. The standard includes a mixture of purified proteins with known molecular weights, allowing users to assess the performance of their analytical equipment.
Automatically generated - may contain errors
Lab products found in correlation
Flexanalysis 3, by Bruker (7 mentions)
Trifluoroacetic acid, by Merck Group (6 mentions)
Peptide calibration standard 2, by Bruker (6 mentions)
α cyano 4 hydroxycynnamic acid, by Merck Group (3 mentions)
Acetonitrile, by Merck Group (3 mentions)
Flexanalysis software, by Bruker (3 mentions)
Sinapinic acid, by Bruker (3 mentions)
Pcr purification kit, by Qiagen (3 mentions)
Taq ligase, by New England Biolabs (3 mentions)
Dntp solutions, by New England Biolabs (3 mentions)
Peptide calibration standard 2 for maldi tof ms, by Bruker (3 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Restriction endonuclease, by New England Biolabs (3 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (3 mentions)
Flexcontrol 3, by Bruker (3 mentions)
Maldi tof target plate, by Bruker (2 mentions)
Microflex lt maldi tof mass spectrometer, by Bruker (2 mentions)
Msp 96 microscout target, by Bruker (2 mentions)
Autoflex 3, by Bruker (2 mentions)
57 protocols using protein calibration standard 1
1
MALDI-TOF MS Protein Extraction
2
Rapid MALDI-TOF Protein Profiling
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Ten µL of crushed abdomens were mixed with 20 µL of 70 % formic acid (v/v) and 20 µL of 50 % acetonitrile (v/v) (Fluka, Buchs, Switzerland) and centrifuged at 10,000 rpm for 20 s. One µL of supernatant of each sample was deposited on the MALDI-TOF target plate in quadruplicate (Bruker Daltonics, Wissembourg, France) and recovered with 1 µL of CHCA matrix solution composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50 % acetonitrile (v/v), 2.5 % trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water. After drying for several minutes at RT, the target was introduced into Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. To control loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I).
3
MALDI-TOF Mass Spectrometry Identification Protocol
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Each colony was placed directly on an individual spot on a 96-spot reusable MALDI-TOF target plate (Bruker Daltonics, Wissembourg, France; cat no. 8280800). Each spot was covered with 1 μl of α-Cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics, Wissembourg, France; cat no. 8255344) diluted in standard solvent (50% acetonitrile: 47.5% water: 2.5% trifluoroacetic acid, Sigma-Aldrich, Lyon, France; cat no. 19182). The matrix was allowed to dry at room temperature, and the target plate was placed in the MALDI Biotyper® with benchtop microflex™ LT/SH mass spectrometer (Bruker Daltonics, Germany). A bacterial test standard (Bruker Protein Calibration Standard I, Bruker Daltonics, Wissembourg, France; cat no. 8255343) was used according to the manufacturer’s instructions to control for loading and matrix. Spectra were compared with the MBT 7854 MSP Library database installed on the computer (Bruker Daltonics, Wissembourg, France; ref no. 182903). An isolate was identified when spectra had a log score value ≥ 1.9 [81 (link)]. Every unidentified isolate was tested successively, where a portion of the same colony was placed on a new spot on the target plate and identified as described.
4
Pellet Characterization of Protein Complexes
5
Metal-Cation Binding to GRN Proteins
6
Mosquito Proteome Profiling with MALDI-TOF MS
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Each whole mosquito specimen was homogenized in 20, 30, 40 and 50 μL of 70% formic acid for stages L2, L3, L4 and pupae, respectively. Homogenizations were performed manually using pestles (Fischer Scientific, Strasbourg, France) or with a FastPrep-24 Instrument (MP BIOMEDICALS, Santa Ana, California, USA) using glass beads (Sigma, Lyon. France). Suspensions were mixed with 50% acetonitrile (v/v) (Fluka, Buchs, Switzerland) and centrifuged at 10,000 rpm for 20 sec. One microliter of the supernatant of each sample was deposited on a steel target plate (Bruker Daltonics, Wissembourg, France) into four spots as previously described [28 (link)]. Then, 1 μL of CHCA matrix composed of saturatedα-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile(v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water was directly overlaid on each spot sample on the target plate, dried for several minutes at room temperature and introduced into the MALDI-TOF MS instrument for analysis. To control loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I).
7
MALDI-TOF Mass Spectrometry for Arthropod Protein Analysis
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On the cut-off legs, a nip of glass powder (Sigma, Lyon, France) was added in addition 30 µl of a mix 70% (v/v) formic acid and 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland) [36] . The legs were crushed and homogenized by a TissueLyserII device (Qiagen, Hilden, Germany) with 30 movements per second for 1 min and repeated three times [45] . After centrifugation of homogenized arthropod legs (at 2000 g for 30 sec), 1 µl of the supernatant of extracted protein was dropped onto a spot of MALDI-TOF polished steel plate (Bruker Daltonic, Wissembourg, France) in quadruplicate [45] . At room temperature, the plate was left to dry and then each spot covered with 1 µl of CHCA matrix solution that composed of saturated αcyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% tri uoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water [41] . After drying, the plate was loaded into the Micro ex LT MALDI-TOF MS apparatus (Bruker Daltonics, Germany) for analysis. Matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I) to control loading on the MS target plate, matrix quality and MALDI-TOF apparatus performance [45] .
8
Mosquito Abdominal Protein Extraction
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A piece of the WFPs (i.e. about 1 mm 2 ) containing crushed abdomens from engorged mosquitoes was individually cut using a sterile scalpel and transferred to a new 1.5 mL Eppendorf tube (Niare et al. 2017) . For each piece of WFPs, 20 µL of formic acid (70%, v/v) plus 20 µL of acetonitrile (50% v/v) (Fluka, Buchs, Switzerland) was added and incubated for 10 min at RT. After a fast spin (i.e. 10 000 rpm for 20 s), 1 µL of the supernatant of each sample was loaded onto the MALDI-TOF target plate in quadruplicate and covered with 1 µL of CHCA matrix (Niare et al. 2016) . After drying for several minutes at RT, the MALDI-TOF target plate was placed in the Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Bremen, Germany) for analysis. To control loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, the matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I) (Niare et al. 2016 ).
9
MALDI-TOF MS for Protein Identity
10
MALDI-TOF Mass Spectrometry of Skimmed Milk
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After thawing, skimmed milk fractions were centrifuged at 20,000 g for 20 min at + 4°C to remove cell debris and bacteria. Then, each sample was diluted 1:100 with ultrapure water (20 (link)) and mixed (1:1) with a matrix solution composed of sinapinic acid (Bruker Daltonics, Bremen, Germany) saturated in 50% v/v acetonitrile containing 0.1% trifluoroacetic acid (Merck Darmstadt, Germany). An aliquot (0.5 μl) of each sample was spotted in triplicate on an MSP 96 target ground steel BC (Bruker Daltonics), previously overlaid with a thin layer of sinapinic acid saturated in 100% ethanol (Merck), and allowed to dry for 10 min at room temperature. Spectral profiles were acquired using a MALDI-TOF Microflex LRF mass spectrometer (Bruker Daltonics) equipped with the FlexControl (v. 3.4) software (Bruker Daltonics). Spectra were recorded in the positive linear ion mode within the mass range 4,000–20,000 m/z (laser frequency 20 Hz; ion source 1 voltage, 19.53 kV; ion source 2 voltage, 18.12 kV; lens voltage, 8.12 kV). Three independent spectra for each sample (500 shots each at random positions on the same target place, for spectrum) were manually collected, externally calibrated by the Protein Calibration Standard 1(Bruker Daltonics) and subsequently analyzed.
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