The SOCS3 mimetic peptides, named KIR or KIR-ESS, were designed starting from structural and biochemical studies of SOCS3/Jak2 complex [18 (link)]. Control peptides (Ctrl1 and Ctrl2) were identical to SOCS3 KIR-ESS region with aminoacid substitutions in positions critical for SOCS3 and Jak2 interactions (Table 1 ). Peptides were synthesized through solid phase peptide synthesis, performed on a fully automated multichannel peptide synthesizer Syro I (Multisynthech, Witten, Germany). Preparative RP-HPLC was carried out on a Shimadzu LC-8A, equipped with a SPD-M10 AV detector and a Phenomenex C18 Jupiter column (50 × 22 mm ID; 10 μm) (Shimadzu, Japan). Peptides were then, analyzed by mass spectrometry, carried out on an LCQ DECA XP Ion Trap mass spectrometer equipped with an OPTON ESI source, operating at 4.2 kV and 320°C, and with a complete Surveyor HPLC system (ThermoFisher Scientific, Waltham, MA USA). To facilitate the peptide delivery into cell cytoplasm, the fragment 48–60 of the HIV Tat protein was conjugated to SOCS3 or to control peptides in a stepwise manner. Purified peptides were lyophilized and stored at −20°C until use.
Lcq deca xp ion trap mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LCQ Deca XP ion-trap mass spectrometer is a versatile analytical instrument designed for qualitative and quantitative analysis of chemical compounds. It utilizes ion-trap technology to capture, isolate, and detect ions based on their mass-to-charge ratio. The LCQ Deca XP is capable of performing a range of mass spectrometry techniques, including full-scan, selected ion monitoring, and tandem mass spectrometry (MS/MS) for structural elucidation.
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Lab products found in correlation
Surveyor hplc system, by Thermo Fisher Scientific (3 mentions)
In line liquid chromatography, by Thermo Fisher Scientific (2 mentions)
Sequest search software, by Thermo Fisher Scientific (2 mentions)
Lc 8a, by Shimadzu (1 mentions)
Xcalibur 2, by Thermo Fisher Scientific (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Aeris widepore 150 2.1 mm, by Phenomenex (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
9 protocols using lcq deca xp ion trap mass spectrometer
1
Synthetic SOCS3 Mimetic Peptides
2
Synthesis and Characterization of Peptides
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Peptide sequences analyzed in this study were synthesized as previously reported [37 (link)], in acetylated and amidated version and are reported in Table 1 . Reagents for chemical synthesis were obtained from Iris Biotech (Marktredwitz, Germany) and solvents were purchased from Romil (Dublin, Ireland). Peptides were treated with HFIP (at 50% (v/v) in water), purified through RP-HPLC and then identified through LC-MS system LCQ DECA XP Ion Trap mass spectrometer from ThermoFisher (Waltham, MA, USA). Purified peptides were lyophilized and stored at −20 °C until use. A small aliquot of Aβ21–40 was oxidized at 1 mg/mL in phosphate buffer (100 mM, pH = 7.2, H2O2 0.1% (v/v)), under stirring for 20 h and then further purified [38 (link)].
The metal compounds (Figure 1 ) were synthetized as previously described [22 (link),39 (link)].
The metal compounds (
3
Identification of Tryptic Peptides by nLC-MS/MS
4
Fingerprinting Metabolites via FIMS-MS
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The FIMS system consisted
of a LCQ DecaXP ion-trap mass spectrometer (Thermo Fisher Scientific
Inc., Waltham, MA, USA) with an Agilent 1200 HPLC system (a quaternary
pump with a vacuum degasser, a thermostated column compartment, an
autosampler, and a diode array detector (DAD) (Agilent Technologies,
Palo Alto, CA, USA). The flow injection was passed through a guard
column (Adsorbosphere All-Guard Cartridge, C18, 5 μm, 4.6 ×
7.5 mm, Alltech Associates, Inc., Deerfield, IL, USA) to minimize
potential contamination for the MS system. Mobile phases consisted
of 0.1% formic acid in H2O (A) and 0.1% formic acid in
acetonitrile (B) with isocratic elution at 60:40 (v/v) at a flow rate
of 0.5 mL/min for 1.5 min. Electrospray ionization (ESI) was performed
in positive ion mode from m/z 100
to 2000 to obtain the FIMS fingerprints. The acquisition mode was
centroid. The following conditions were used for the DecaXP mass spectrometer:
sheath gas flow rate, 80 (arbitrary units); auxiliary gas flow rate,
10 (arbitrary units); spray voltage, 4.50 kV; heated capillary temperature,
220 °C; capillary voltage, 4.0 V; tube lens offset, 25 V. Spectra
were summed from 0.4 to 1.0 min interval required for the sample bolus
to enter the MS. Three repeat analyses of the 54 different samples
provided 162 spectra.
of a LCQ DecaXP ion-trap mass spectrometer (Thermo Fisher Scientific
Inc., Waltham, MA, USA) with an Agilent 1200 HPLC system (a quaternary
pump with a vacuum degasser, a thermostated column compartment, an
autosampler, and a diode array detector (DAD) (Agilent Technologies,
Palo Alto, CA, USA). The flow injection was passed through a guard
column (Adsorbosphere All-Guard Cartridge, C18, 5 μm, 4.6 ×
7.5 mm, Alltech Associates, Inc., Deerfield, IL, USA) to minimize
potential contamination for the MS system. Mobile phases consisted
of 0.1% formic acid in H2O (A) and 0.1% formic acid in
acetonitrile (B) with isocratic elution at 60:40 (v/v) at a flow rate
of 0.5 mL/min for 1.5 min. Electrospray ionization (ESI) was performed
in positive ion mode from m/z 100
to 2000 to obtain the FIMS fingerprints. The acquisition mode was
centroid. The following conditions were used for the DecaXP mass spectrometer:
sheath gas flow rate, 80 (arbitrary units); auxiliary gas flow rate,
10 (arbitrary units); spray voltage, 4.50 kV; heated capillary temperature,
220 °C; capillary voltage, 4.0 V; tube lens offset, 25 V. Spectra
were summed from 0.4 to 1.0 min interval required for the sample bolus
to enter the MS. Three repeat analyses of the 54 different samples
provided 162 spectra.
5
Mass Spectrometry Analysis of Collagen
6
Protein Identification by Mass Spectrometry
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Protein bands were cut from SDS PAGE gels and subjected to in-gel trypsin digestion (39 (link), 67 (link)). Electrospray MS was performed on the tryptic peptides using an LCQ Deca XP ion-trap mass spectrometer equipped with in-line liquid chromatography (Thermo Finnigan LLC) using a C8 capillary column (300 μm × 150 mm; Grace Vydac 208MS5.315) eluted at 4.5 μl/min. Seaquest search software (Thermo Finnigan LLC) was used for peptide identification using the NCBI protein database.
7
Mass Spectrometry Analysis of Collagen
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For mass spectrometry, we prepared type I collagen from Crtap−/− and wildtype tibiae. We defatted bone with chloroform/methanol (3:1 v/v) and demineralized it in 0.5 M EDTA, 0.05 M Tris-HCl, pH 7.5, all steps at 4°C. We finely minced the bone samples and solubilized collagen by heat denaturation (90°C) in SDS-PAGE sample buffer. Collagen α-chains were cut from SDS-PAGE gels and subjected to in-gel trypsin digestion. We performed electrospray MS on the tryptic peptides using an LCQ Deca XP ion-trap mass spectrometer equipped with in-line liquid chromatography (LC) (ThermoFinnigan) using a C8 capillary column (300 μm × 150 mm; Grace Vydac 208 MS5.315) eluted at 4.5 μl min. Sequest search software (ThermoFinnigan) was used for peptide identification using the NCBI protein database.
We quantified pyridinoline cross-links (HP and LP) by HPLC after hydrolyzing demineralized bone in 6 N HCl as described45 (link).
We quantified pyridinoline cross-links (HP and LP) by HPLC after hydrolyzing demineralized bone in 6 N HCl as described45 (link).
8
Synthesis and Characterization of Lipidated Lewis Y
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Lewis Y (LeY)-hexadecanohydrazide (palmitic hydrazide) ea. palmitoylated LeY (from here on, shortly, lipo-LeY) was prepared from LeY pentasaccharide (Elicityl, Crolles, France) and hexadecanohydrazide through a reductive amination reaction. Addition of CHCl3/MeOH/H2O at 8:1:8 v/v ratio, followed by vigorous stirring and centrifugation, allowed the extraction of lipo-LeY as a white slurry at the interphase. The slurry was freeze-dried to remove residual solvent. The correct mass of the glycolipid was confirmed by ESI-MS (Thermo Finnigan LCQ-Deca XP Iontrap mass spectrometer in positive mode) using nanospray capillary needle.
9
Quantitative Analysis of WNK1 Autophosphorylation
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