The chemokines CXCL9, CXCL10 or CXCL11 (20 µg/mL) were incubated with 12.5 units per liter (U/L) natural human CD26, with or without 0.88 to 26.4 µg/mL heparin, heparin DP8, heparin DP30, heparan sulfate, dermatan sulfate (Iduron, Chechire, U.K.), chondroitin sulfate A or chondroitin sulfate C (Sigma-Aldrich) in 50 mM EDTA; 1 mM Tris buffer (pH 7.5) in a total volume of 25 µL in low-binding tubes (Eppendorf LoBind Tube 1,5 mL, Eppendorf AG, Hamburg, Germany). An overview of the GAGs used in experiments is provided in Table 2 . Nota bene, “DP” refers to the number of disaccharides. After 2 h of incubation, enzymatic reactions were terminated by acidification up to 0.08% (v/v) TFA. Chemokines were extracted and desalted from total samples on C18 ZipTip pipet tips (Millipore Corporation), eluted with 50% (v/v) acetonitrile in 0.1% (v/v) TFA, and analyzed by mass spectrometry.
Ziptip c18 pipet tips
Manufactured by Merck Group
Sourced in United States
The ZipTip C18 pipet tips are designed for desalting, purification, and sample preparation in analytical applications. They are made of a hydrophobic polystyrene-divinylbenzene (PS-DVB) polymer matrix with a C18 functional group. The ZipTip C18 tips can be used to selectively capture and concentrate analytes from complex samples prior to analysis by techniques such as mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Chondroitin sulfate c, by Merck Group (1 mentions)
Chondroitin sulfate a, by Merck Group (1 mentions)
Proteasemax, by Promega (1 mentions)
Proteome discoverer 2, by Matrix Science (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Spectramax gemini em, by Molecular Devices (1 mentions)
Ultimate 3000 nano hplc system, by Thermo Fisher Scientific (1 mentions)
Nanospray flex, by Thermo Fisher Scientific (1 mentions)
4800 plus maldi tof toftm analyzer, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
9 protocols using ziptip c18 pipet tips
1
CXCL9/10/11 Chemokine Modulation by Glycosaminoglycans
2
Proteomic Analysis of Murine Islets
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Murine islets were prepared for proteomics and run on a Q Exactive Orbitrap mass spectrometer (ThermoScientific), following our optimized protocol for accurate detection of deamidated peptides, including manual inspection of spectra, as previously described (26 (link)). Briefly, cell lysates were reduced, alkylated and quenched, followed by protein precipitation using the Wessel-Flügge method. Digestion was performed in ammonium bicarbonate pH 8 with modified trypsin (Pierce; protein/trypsin ratio 1:20 (w/w)) for 90 minutes at 37°C in the presence of 5% acetonitrile and 0.01% ProteaseMAX (Promega). Peptide mixtures were subjected to desalting with C18 ZipTip pipet tips (Millipore) and loaded on an Ultimate 3000 UPLC system (Dionex, Thermo Scientific) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). Peptides were identified by Mascot (Matrix Science) using SwissProt as a database through Proteome Discoverer 2.2, incorporating Percolator for peptide validation. Two missed cleavages were allowed, peptide tolerance was set at 5 ppm, and MS/MS tolerance at 20 mmu. Carbamidomethylation (C) was included as a fixed modification and oxidation (M) and deamidation (N/Q) were included as variable modifications.
3
Nano-LC-MS Proteomics Analysis Protocol
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Samples were purified by ZipTip C18 pipet tips
(Millipore) according to the manufacturer’s instructions prior
to nanoLC-ESI-MS analysis. Purified samples were analyzed using an
UltiMate 3000 nano-HPLC system coupled to a Q Exactive HF mass spectrometer
(both Thermo Scientific, Bremen, Germany) equipped with a Nanospray
Flex ionization source. The samples were separated on a homemade fritless
fused-silica microcapillary column (75 μm i.d. × 280 μm
o.d. × 10 cm length) packed with 3 μm reversed-phase C18
material (Reprosil). Solvents for HPLC were 0.1% formic acid (solvent
A) and 0.1% formic acid in 85% acetonitrile (solvent B). The gradient
profile was as follows: 0–2 min, 4% B; 2–40 min, 4–50%
B; 40–45 min, 50–100% B, and 45–50 min, 100%
B. The flow rate was 250 nL/min. The Q Exacitve HF mass spectrometer
was operating in the data dependent mode selecting the top 20 most
abundant isotope patterns with charge >1 from the survey scan with
an isolation window of 1.6 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired from
300 to 1750 m/z at a resolution
of 60,000 with a maximum injection time (IT) of 120 ms, and automatic
gain control (AGC) target 1e6. The selected isotope patterns were
fragmented by higher-energy collisional dissociation (HCD) with normalized
collision energy of 28 at a resolution of 30,000 with a maximum IT
of 120 ms, and AGC target 5e5.
(Millipore) according to the manufacturer’s instructions prior
to nanoLC-ESI-MS analysis. Purified samples were analyzed using an
UltiMate 3000 nano-HPLC system coupled to a Q Exactive HF mass spectrometer
(both Thermo Scientific, Bremen, Germany) equipped with a Nanospray
Flex ionization source. The samples were separated on a homemade fritless
fused-silica microcapillary column (75 μm i.d. × 280 μm
o.d. × 10 cm length) packed with 3 μm reversed-phase C18
material (Reprosil). Solvents for HPLC were 0.1% formic acid (solvent
A) and 0.1% formic acid in 85% acetonitrile (solvent B). The gradient
profile was as follows: 0–2 min, 4% B; 2–40 min, 4–50%
B; 40–45 min, 50–100% B, and 45–50 min, 100%
B. The flow rate was 250 nL/min. The Q Exacitve HF mass spectrometer
was operating in the data dependent mode selecting the top 20 most
abundant isotope patterns with charge >1 from the survey scan with
an isolation window of 1.6 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired from
300 to 1750 m/z at a resolution
of 60,000 with a maximum injection time (IT) of 120 ms, and automatic
gain control (AGC) target 1e6. The selected isotope patterns were
fragmented by higher-energy collisional dissociation (HCD) with normalized
collision energy of 28 at a resolution of 30,000 with a maximum IT
of 120 ms, and AGC target 5e5.
4
Malachite Green Aptamer Binding Assay
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The binding of malachite green
to the aptamer was measured with a SpectraMAX GeminiEM fluorescence
plate reader (Molecular Devices) with excitation wavelength 610 nm
and emission wavelength 670 nm. The malachite green stock solution
was mixed with RNA top and bottom strands to the final concentration
of 2 μM malachite green and 1 μM of each strand of the
aptamer in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl.
In RNA primer
extension reactions, the RNA strands were mixed with 2-MeImpG to final
concentrations of 50 mM 2-MeImpG and 5 μM each RNA strand (i.e.,
5 μM of the RNA complex) in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl,
50 mM MgCl2. After the reaction, samples were desalted
using ZipTip C18 pipet tips (Millipore). Samples were eluted
from the tip with 1:1 acetonitrile:water, solvent was removed on a
SpeedVac vacuum concentrator, and the RNA was dissolved in water.
The concentration of the sample was measured using a NanoDrop spectrophotometer,
adjusted to 2 μM of the RNA complex, and analyzed as shown above.
to the aptamer was measured with a SpectraMAX GeminiEM fluorescence
plate reader (Molecular Devices) with excitation wavelength 610 nm
and emission wavelength 670 nm. The malachite green stock solution
was mixed with RNA top and bottom strands to the final concentration
of 2 μM malachite green and 1 μM of each strand of the
aptamer in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl.
In RNA primer
extension reactions, the RNA strands were mixed with 2-MeImpG to final
concentrations of 50 mM 2-MeImpG and 5 μM each RNA strand (i.e.,
5 μM of the RNA complex) in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl,
50 mM MgCl2. After the reaction, samples were desalted
using ZipTip C18 pipet tips (Millipore). Samples were eluted
from the tip with 1:1 acetonitrile:water, solvent was removed on a
SpeedVac vacuum concentrator, and the RNA was dissolved in water.
The concentration of the sample was measured using a NanoDrop spectrophotometer,
adjusted to 2 μM of the RNA complex, and analyzed as shown above.
5
Nanoflow LC-MS/MS Proteomics Analysis
6
Non-enzymatic RNA Polymerization Protocol
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Non-enzymatic
RNA polymerization was initiated by addition of activated
RNA monomer(s) to the mixture of 3′-truncated and template
oligonucleotides. Specific reaction conditions are given below for
each functional RNA system. All reaction mixtures were incubated at
room temperature for specified times, desalted using ZipTip C18 pipet tips (Millipore), and analyzed by TBE–urea
20% PAGE.
RNA polymerization was initiated by addition of activated
RNA monomer(s) to the mixture of 3′-truncated and template
oligonucleotides. Specific reaction conditions are given below for
each functional RNA system. All reaction mixtures were incubated at
room temperature for specified times, desalted using ZipTip C18 pipet tips (Millipore), and analyzed by TBE–urea
20% PAGE.
7
Protein Digestion and MALDI-TOF/MS Analysis
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Protein digestion and MALDI-TOF/MS analyses were performed as follows. Each protein gel piece was destained with 100 mM NH4HCO3 in 30% (v/v) acetonitrile (ACN) for 2 h at 40 °C. The gel pieces were minced, lyophilized and digested in 25 mM NH4HCO3 with 10 ng sequencing-grade modified trypsin (Promega, Madison, WI, USA) at 37 °C overnight. After digestion, peptides were extracted by three washes with 0.1% trifluoroacetic acid (TFA) in 60% ACN. The peptides were desalted by ZipTipC-18 pipet tips (Millipore, Bedford, MA, USA) according to the product manual. Tryptic peptide samples were analyzed using a 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, Foster, CA, USA).
8
Hammerhead Ribozyme Reaction Protocol
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Hammerhead ribozyme reactions were
initiated by mixing both RNA strands to a final concentration of 2.5
μM each strand, in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl, 50 mM
MgCl2. Reactions where primer extension was performed contained
50 mM 2-MeImpG. Reactions were incubated at room temperature for 24
h. After the reaction, samples were desalted using ZipTip C18 pipet tips (Millipore) and analyzed by TBE–urea 20% PAGE.
The reaction yield was calculated as the percentage of the product
band intensity relative to the total intensity of all bands on the
gel.
initiated by mixing both RNA strands to a final concentration of 2.5
μM each strand, in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl, 50 mM
MgCl2. Reactions where primer extension was performed contained
50 mM 2-MeImpG. Reactions were incubated at room temperature for 24
h. After the reaction, samples were desalted using ZipTip C18 pipet tips (Millipore) and analyzed by TBE–urea 20% PAGE.
The reaction yield was calculated as the percentage of the product
band intensity relative to the total intensity of all bands on the
gel.
9
Ribozyme-Catalyzed Diels-Alderase Reaction
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The Diels–Alderase
reaction was initiated by mixing 9-hydroxymethylanthracene
and N-propylmaleimide to final concentrations of
200 μM 9-hydroxymethylanthracene and 1000 μM N-propylmaleimide, in100 mM Tris-HCl pH 7.5, 100 mM MgCl2, and 300 mM NaCl. The Diels–Alderase ribozyme-catalyzed
reactions were performed in the presence of 10 μM RNA. In RNA
primer extension reactions, the RNA strands were mixed with 2-MeImpG
to final concentrations of 50 mM 2-MeImpG and 5 μM each RNA
strand in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl, 50 mM MgCl2. Reactions were incubated at room temperature for 24 h. After the
reaction, samples were desalted using ZipTip C18 pipet
tips (Millipore). Samples were eluted from the tip with 1:1 acetonitrile:water,
solvent was removed on a SpeedVac vacuum concentrator, and the RNA
was dissolved in water. The concentration of the sample was measured
using a NanoDrop spectrophotometer, the concentration was adjusted
to 10 μM of the RNA complex, and this solution was subjected
to the above ribozyme reaction conditions.
The reactions were
incubated at room temperature for the reported amount of time and
analyzed on an Agilent 1100 Analytical HPLC with a Varian Microsorb-mv
100-8 C18 250 × 4.6 mm column with a gradient of solvent
A: 0.1% TFA in H2O, and solvent: B 0.1% TFA in acetonitrile,
with UV detection at 260 nm.
reaction was initiated by mixing 9-hydroxymethylanthracene
and N-propylmaleimide to final concentrations of
200 μM 9-hydroxymethylanthracene and 1000 μM N-propylmaleimide, in100 mM Tris-HCl pH 7.5, 100 mM MgCl2, and 300 mM NaCl. The Diels–Alderase ribozyme-catalyzed
reactions were performed in the presence of 10 μM RNA. In RNA
primer extension reactions, the RNA strands were mixed with 2-MeImpG
to final concentrations of 50 mM 2-MeImpG and 5 μM each RNA
strand in 0.25 M Tris-HCl pH 8.0, 0.15 M NaCl, 50 mM MgCl2. Reactions were incubated at room temperature for 24 h. After the
reaction, samples were desalted using ZipTip C18 pipet
tips (Millipore). Samples were eluted from the tip with 1:1 acetonitrile:water,
solvent was removed on a SpeedVac vacuum concentrator, and the RNA
was dissolved in water. The concentration of the sample was measured
using a NanoDrop spectrophotometer, the concentration was adjusted
to 10 μM of the RNA complex, and this solution was subjected
to the above ribozyme reaction conditions.
The reactions were
incubated at room temperature for the reported amount of time and
analyzed on an Agilent 1100 Analytical HPLC with a Varian Microsorb-mv
100-8 C18 250 × 4.6 mm column with a gradient of solvent
A: 0.1% TFA in H2O, and solvent: B 0.1% TFA in acetonitrile,
with UV detection at 260 nm.
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