Purified Dyrk2 was cross-linked at a concentration of 0.5 mg/ml with 1 mM isotope labeled di-succinimidylsuberate (DSS-d0, DSS-d12) (CreativeMolecules Inc.) at 37 °C for 30 min as previously described28 (link). The reaction was quenched with 100 mM NH4CO3 for 30 min. Afterwards, the sample was dried in a speedvac, re-dissolved in 8 M Urea, reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 10 mM iodacetamide. For digestion, the sample was diluted to 1 M urea and trypsin (protease/protein ratio 1:50) was added over night. Digestion was stopped by acidification with 5% formic acid and peptides were purified by C18 clean-up. Dried peptides were dissolved in 20 μl 0.1% formic acid and 30% acetonitrile. Cross-linked peptides were enriched by peptide size-exclusion chromatography with Superdex Peptide PC 3.2/30 column (GE Healthcare, Uppsala) using running buffer containing 30% acetonitrile and 0.1% formic acid. SEC fractions were then dissolved in 5% acetonitrile and 0.1% formic acid, iRT peptides (Biognosys) were spiked to each sample before LC–MS/MS analysis for quality control and retention time alignment.
Superdex peptide pc 3.2 30 column
Manufactured by GE Healthcare
Sourced in Japan, Spain
The Superdex Peptide PC 3.2/30 column is a size exclusion chromatography column designed for the analysis and purification of peptides. It has a column volume of 2.4 ml and is compatible with a variety of aqueous and organic solvents. The column is suitable for the separation of peptides based on their molecular size.
Automatically generated - may contain errors
Lab products found in correlation
12 t solarix hybrid fourier transform ion cyclotron resonance ft icr mass spectrometer, by Bruker (2 mentions)
Model spe ma10avp, by Shimadzu (2 mentions)
Spe ma10avp, by Shimadzu (2 mentions)
Irt peptide, by Biognosys (1 mentions)
Rapigest, by Waters Corporation (1 mentions)
1200 infinity hplc system, by Agilent Technologies (1 mentions)
Oasis hlb elution plate, by Waters Corporation (1 mentions)
Benzamidine hydrochloride, by Merck Group (1 mentions)
Milli q water, by Merck Group (1 mentions)
17 protocols using superdex peptide pc 3.2 30 column
1
Cross-linking and Mass Spectrometry of Dyrk2
2
Glycan Sequencing Using PRAGS Labeling
3
Molecular Weight Distribution Analysis by SEC
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Size exclusion chromatography (SEC) was utilized to obtain the molecular weight distribution of the hydrolysates, following the protocol of Martínez-Alvarez et al. [68 (link)]. SEC was carried out using an HPLC model LC-10ADVP (Shimadzu, Tokyo, Japan) and a Superdex Peptide PC 3.2/30 column (GE Healthcare, Barcelona, Spain) with a fractionation range between 7000–100 Da. The solvent utilized was 30% (v/v) acetonitrile containing 0.1% (v/v) TFA, at 25 °C, with a flow rate of 0.1 mL/min. Bovine serum albumin was used to determine the void volume, while aprotinin, angiotensin II, vitamin B12, N-hippuryl-His-leucine (HHL) and glycine were used as standards. Absorbance readings were taken at wavelengths of 214 and 280 nm.
4
Heparan Sulfate Digestion and Profiling
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Digestion of porcine intestinal mucosa heparan sulfate (350 µg) was performed in a 1 ml solution system with 500 µl digestion buffer (100 mM NaCl, 20 mM Tris-HCl, 1 mM Ca(OAc)2, pH 7.4) at 37 °C. An aliquot of 50 mIU of a single heparin lyase enzyme (lyase I or lyase III) was added for overnight for complete digestion. The digestion was stopped by heating at 100 °C for 10 min. The digestion products were dried by centrifugal evaporation, reconstituted in water and purified/profiled using a Superdex™ peptide PC 3.2/30 column (GE healthcare). The column was equilibrated and operated using a 50 mM ammonium acetate buffer in 10% acetonitrile. The fraction corresponding to DP4 (DP: degree of polymerization), DP6 and DP8 oligosaccharides were collected for HSulf2 treatment and further MS analysis.
5
Enoxaparin Tetrasaccharide Isolation
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The enoxaparin was obtained from
the Lovenox injection syringe.
A total of ∼100 μg of enoxaparin was digested by a mixture
of heparin lyase I, II, and III (10 mU each) in a digestion buffer
consisting of 50 mM NaOAc, pH 6.0, 1 mM Ca(OAc)2, and 0.1%
BSA. After incubation at 37 °C overnight, the digestion products
were fractionated using a Superdex Peptide PC 3.2/30 column (GE Biosciences,
Piscataway, NJ) equilibrated with 100 mM NH4HCO3. The chromatographic peak corresponding to tetrasaccharide was collected
as the lyase-resistant tetrasaccharides and vacuum-dried immediately
to avoid unwanted degradation.
the Lovenox injection syringe.
A total of ∼100 μg of enoxaparin was digested by a mixture
of heparin lyase I, II, and III (10 mU each) in a digestion buffer
consisting of 50 mM NaOAc, pH 6.0, 1 mM Ca(OAc)2, and 0.1%
BSA. After incubation at 37 °C overnight, the digestion products
were fractionated using a Superdex Peptide PC 3.2/30 column (GE Biosciences,
Piscataway, NJ) equilibrated with 100 mM NH4HCO3. The chromatographic peak corresponding to tetrasaccharide was collected
as the lyase-resistant tetrasaccharides and vacuum-dried immediately
to avoid unwanted degradation.
6
Cross-linking and Fractionation of CSN-CRL2 Complexes
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Twenty microlitres of ~20 μM CSN–CRL2~N8, CSNWT–CRL2 and apo-CSNWT were each incubated with 1–5 mM BS3 cross-linker for 1 h at 25 °C and 350 r.p.m. in a thermomixer. After cross-linking, complexes were (i) separated by gel electrophoresis (NuPAGE) followed by in-gel digestion (CSNWT–CRL2, CSNWT and CSN–CRL2~N8) or (ii) digested in solution (CSNWT–CRL2) and generated peptides were pre-fractionated by gel filtration (CSN–CRL2~N8). Gel electrophoresis was performed using the NuPAGE system according to manufacturer’s protocols. In-gel digestion was performed as described before51 (link). Digestion in solution was performed in the presence of RapiGest (Waters) according to manufacturer’s protocols. For gel filtration, peptides were dissolved in 30% acetonitrile (ACN), 0.1% trifluoroacetic acid and separated on a Superdex Peptide PC 3.2/30 column (GE Healthcare) at a flow rate of 50 µl min−1.
7
SEC-MS Analysis of Heparin Lyase Digests
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Heparin lyase digestion products ranging from disaccharide to tetrasaccharide, were directly analyzed using SEC-MS as described previously. [45] , [52] (link) Briefly, HS samples (∼100 pmol) were injected onto a Superdex Peptide PC 3.2/30 column (GE Biosciences, Piscataway, NJ) online with an Applied Biosystems/MDS Sciex QSTAR Pulsar Qq-TOF mass spectrometer operating in the negative-ion mode. The isocratic mobile phase contains 12.5 mM formic acid, titrated to pH 4.4 with ammonia, in 10% acetonitrile.
8
Quantitative Peptide Profiling using SPE
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BSA (A7030) and BS3 d0/d12 were
purchased from Sigma-Aldrich (St. Louis, MO) and Creative Molecule
Inc., respectively. Rapigest and solid-phase extraction (SPE) cartridges
(50 mg Sep-Pak C18) were purchased from Waters Corporation (Milford,
MA, U.S.A.). The Superdex Peptide PC 3.2/3.0 column was purchased
from GE Healthcare (Piscataway, NJ).
purchased from Sigma-Aldrich (St. Louis, MO) and Creative Molecule
Inc., respectively. Rapigest and solid-phase extraction (SPE) cartridges
(50 mg Sep-Pak C18) were purchased from Waters Corporation (Milford,
MA, U.S.A.). The Superdex Peptide PC 3.2/3.0 column was purchased
from GE Healthcare (Piscataway, NJ).
9
Oligosaccharide Sequencing via PRAGS
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Purified oligosaccharide products were labeled with O-(pyridin-3-ylmethyl)hydroxylamine, a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS), as previously described.15 (link) Briefly, about 1 nmol of dried oligosaccharide products were dissolved in 12 μL 1% acetic acid and mixed with 3 μL of a 30 mM solution of PRAGS reagent in acetonitrile. After incubation at 37 °C for overnight (∼16 h), the labeled oligosaccharides were separated from the remaining excess reagent and salts on a Superdex Peptide PC 3.2/30 column (GE Biosciences, Piscataway, NJ) equilibrated with 25 mM ammonium acetate, 5% acetonitrile.
The desalted oligosaccharides were dissolved in 5% isopropanol, 0.1% ammonia to a final concentration of 10 pmol μL–1 and directly infused into a 12 T solariX™ hybrid Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Bremen, Germany) using an Apollo II nanoESI source. The instrument was operated in the negative mode and precursor ions of interest were isolated with an isolation window of 7 Th. Collision induced dissociation (CID) was carried out in the hexapole collision cell.
The desalted oligosaccharides were dissolved in 5% isopropanol, 0.1% ammonia to a final concentration of 10 pmol μL–1 and directly infused into a 12 T solariX™ hybrid Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Bremen, Germany) using an Apollo II nanoESI source. The instrument was operated in the negative mode and precursor ions of interest were isolated with an isolation window of 7 Th. Collision induced dissociation (CID) was carried out in the hexapole collision cell.
10
Cross-Linking Mass Spectrometry Workflow
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For cross-linking mass spectrometry, digested peptides were concentrated and desalted using an OASIS® HLB µElution Plate (Waters) according to manufacturer instructions. Cross-linked peptides were enriched using size exclusion chromatography (Leitner et al, 2012 (link)). In brief, desalted peptides were reconstituted with SEC buffer (30% (v/v) ACN in 0.1% (v/v) TFA) and fractionated using a Superdex Peptide PC 3.2/30 column (GE) on a 1200 Infinity HPLC system (Agilent) at a flow rate of 0.05 mL/min. Fractions eluting between 50–70 µl were evaporated to dryness and reconstituted in 30 μl 4% (v/v) ACN in 1% (v/v) FA.
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