The protein digestion was then fractionated by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 8 over 80 min into 80 fractions. Then, the peptides were combined into 18 fractions for the global proteome analysis as previously reported [13 (link)]. The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifuging.
Agilent 300 extend c18 column
Manufactured by Agilent Technologies
Sourced in United States, China
The Agilent 300 Extend C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with chemically bonded C18 alkyl chains, providing reversed-phase chromatographic separation.
Automatically generated - may contain errors
Lab products found in correlation
Strata x c18 spe column, by Phenomenex (19 mentions)
C18 ziptip, by Merck Group (10 mentions)
Agilent 1100 binary pump, by Agilent Technologies (6 mentions)
Q exactive plus, by Thermo Fisher Scientific (5 mentions)
Tmt kit, by Thermo Fisher Scientific (4 mentions)
Six plex tmt kit, by Thermo Fisher Scientific (4 mentions)
Agilent 1260 binary pump, by Agilent Technologies (4 mentions)
Trypsin, by Promega (3 mentions)
Easy nlc 1000 uplc system, by Thermo Fisher Scientific (3 mentions)
Strata x c18, by Phenomenex (3 mentions)
Photodiode array detector, by Thermo Fisher Scientific (3 mentions)
Tmt reagent, by Thermo Fisher Scientific (3 mentions)
8 plex itraq kit, by Merck Group (2 mentions)
High intensity ultrasonic processor, by Ningbo Scientz Biotechnology (2 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (2 mentions)
Antibody beads, by PTM Biolabs (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
1200 pump, by Agilent Technologies (1 mentions)
101 protocols using agilent 300 extend c18 column
1
High-pH Reverse-Phase HPLC Fractionation
2
High-pH HPLC Fractionation and Lysine Succinylation Enrichment
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The tryptic peptides were then fractionated into several fractions by high-pH reverse-phase HPLC using Agilent 300 Extend C18 columns (5 μm particles, 4.6 mm ID, 250 mm length). Briefly speaking, peptides were first separated into 60 fractions with a gradient of 8%–32% acetonitrile (ACN, pH 9.0) for over 60 min. Afterwards, the peptide fractions were combined into 4 fractions and dried by vacuum centrifuging.
Enrichment was implemented by immunoprecipitation in accordance with previous studies [28 (link), 29 (link)]. Briefly, to enrich lysine-succinylation modified peptides, tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, and pH 8.0) and then incubated overnight with prewashed antisuccinyl lysine antibody agarose beads (catlog no. PTM402; PTM Bio, Hangzhou, China) at 4°C with gentle shaking. Finally, the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA), combined, and vacuum-dried. Before LC-MS/MS analysis, the obtained peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions.
Enrichment was implemented by immunoprecipitation in accordance with previous studies [28 (link), 29 (link)]. Briefly, to enrich lysine-succinylation modified peptides, tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, and pH 8.0) and then incubated overnight with prewashed antisuccinyl lysine antibody agarose beads (catlog no. PTM402; PTM Bio, Hangzhou, China) at 4°C with gentle shaking. Finally, the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA), combined, and vacuum-dried. Before LC-MS/MS analysis, the obtained peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions.
3
High-pH HPLC Peptide Fractionation
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The labeled peptides were fractionated by high pH reverse-phase HPLC using Agilent 300 Extend C18 columns (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, the peptides were initially separated on a 2% to 60% acetonitrile gradient in 10 mM ammonium bicarbonate at pH 10 over 80 min into 80 fractions. The peptides were then combined as per the manufacturer’s protocol for LC-MS/MS analysis.
4
Quantitative Proteomics with TMT Labeling
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After trypsin digestion was completed, peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. Peptides were reconstituted in 0.5 M triethylammonium bicarbonate and labeled. In brief, one unit of TMT reagent (labeled 100 μg of protein) was thawed and reconstituted in 24 μl acetonitrile. The peptide mixtures were incubated for 2 h at room temperature, pooled, desalted, and dried through vacuum centrifugation. Five samples with three biological replicates were labeled with TMT tags. WH100 and WH98 were labeled with 130 and 129 respectively. WH50, WH20, and WH01 were labeled with 128, 127 and 126respectively.
The TMT labeled samples were then fractionated through high pH reverse-phase high-performance liquid chromatography (LC) by using Agilent 300 Extend C18 columns (5 μm particles, 4.6 mm ID, 250 mm length). The LC gradient was run with 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) for 80 min to generate 80 fractions. Afterward, which all of the fractions were combined into 18 fractions. The fractionated samples were dried through vacuum centrifugation and stored at -20°C.
The TMT labeled samples were then fractionated through high pH reverse-phase high-performance liquid chromatography (LC) by using Agilent 300 Extend C18 columns (5 μm particles, 4.6 mm ID, 250 mm length). The LC gradient was run with 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) for 80 min to generate 80 fractions. Afterward, which all of the fractions were combined into 18 fractions. The fractionated samples were dried through vacuum centrifugation and stored at -20°C.
5
Peptide Fractionation and Mass Spectrometry
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Samples were combined 1:1 such that each channel consisted of the same amount of peptide. The pooled peptide sample was desalted with a 100-mg Sep-Pak solid phase extraction column and then fractionated with basic pH reversed-phase (BPRP) HPLC. Fractionation was executed using an Agilent 1200 pump with an Agilent 300 Extend C18 column (3.5-μm particles, inner diameter of 2.1 mm and length of 250 mm). A 50-min linear gradient from 5% to 35% ACN in 10 mM ammonium bicarbonate pH 8 at a column flow rate of 0.25 ml min−1 was used for peptide fractionation. A total of 96 fractions were collected and then concatenated down to 24 superfractions, as described previously69 (link). These 24 superfractions were divided into two sets of 12 non-adjacent superfractions and were acidified by adding formic acid to a concentration of 1%. One set of fractions (n = 12) were vacuum-centrifuged to near dryness, and each was desalted via StageTip, dried by vacuum centrifugation, and reconstituted in 5% acetonitrile, 5% formic acid before LC-MS/MS analysis.
6
Peptide Separation and Analysis
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Peptides were separated
by basic pH reverse-phase HPLC. Samples were solubilized in buffer
A (5% ACN, 10 mM ammonium bicarbonate, and pH 8.0) and subjected to
a 50 min linear gradient from 18 to 45% ACN in 10 mM ammonium bicarbonate
pH 8 at flow rate of 0.8 mL/min. An Agilent 1100 pump equipped with
a degasser and a photodiode array detector was used with an Agilent
300 extend C18 column (5 μm particles, 4.6 mm inner diameter,
and 20 cm length). The peptide mixture was then fractionated into
96 fractions and consolidated into 24 fractions. Samples were acidified
with 10% formic acid and vacuum-dried, followed by redissolving with
5% formic acid/5% ACN for LC–MS/MS processing.
by basic pH reverse-phase HPLC. Samples were solubilized in buffer
A (5% ACN, 10 mM ammonium bicarbonate, and pH 8.0) and subjected to
a 50 min linear gradient from 18 to 45% ACN in 10 mM ammonium bicarbonate
pH 8 at flow rate of 0.8 mL/min. An Agilent 1100 pump equipped with
a degasser and a photodiode array detector was used with an Agilent
300 extend C18 column (5 μm particles, 4.6 mm inner diameter,
and 20 cm length). The peptide mixture was then fractionated into
96 fractions and consolidated into 24 fractions. Samples were acidified
with 10% formic acid and vacuum-dried, followed by redissolving with
5% formic acid/5% ACN for LC–MS/MS processing.
7
High-pH Reverse-Phase Fractionation and Kbhb Enrichment
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We used HPLC buffer A (10 mM ammonium formate in water, pH 7.8) and HPLC buffer B (10 mM ammonium formate in 90% acetonitrile and 10% water, pH 7.8) for high-pH reverse-phase fractionation of peptides. The reverse-phase Agilent 300 Extend C18 column [5-μm particles, 4.6 mm inner diameter, and 250 mm length] was equilibrated with 2% HPLC buffer B. The peptide mixture of interest in HPLC buffer A was loaded onto the HPLC column and eluted with linear gradient of 2 to 30% HPLC buffer B in 60 min, 30 to 90% buffer B in 15 min, and 90% buffer B in 15 min. Eighty-one fractions were collected and combined into 27 final fractions over equal time intervals, followed by lyophilization using SpeedVac.
Enrichment of Kbhb peptides from different fractions by immunoprecipitation with pan anti-Kbhb antibody was carried out as described previously (54 (link)). Briefly, the peptides were first dissolved in NETN buffer [100 mM NaCl, 50 mM tris-HCl, 1 mM EDTA, and 0.5% NP-40 (pH 8.0)] and incubated with pan anti-Kbhb beads at 4°C overnight. Then, the beads were washed three times with NETN buffer and twice with ddH2O. The combined peptides were eluted with 0.1% (v/v) TFA. The isolated Kbhb peptides were dried in SpeedVac.
Enrichment of Kbhb peptides from different fractions by immunoprecipitation with pan anti-Kbhb antibody was carried out as described previously (54 (link)). Briefly, the peptides were first dissolved in NETN buffer [100 mM NaCl, 50 mM tris-HCl, 1 mM EDTA, and 0.5% NP-40 (pH 8.0)] and incubated with pan anti-Kbhb beads at 4°C overnight. Then, the beads were washed three times with NETN buffer and twice with ddH2O. The combined peptides were eluted with 0.1% (v/v) TFA. The isolated Kbhb peptides were dried in SpeedVac.
8
iTRAQ-Based Proteomic and Phosphoproteomic Analysis
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The trypsin-digested protein samples were desalted by filtration through a Strata X C18 SPE column (Phenomenex, Guangzhou, China), followed by vacuum drying. The resultant peptides were reconstituted in 0.5 M TEAB and treated with an 8-plex iTRAQ kit (Sigma) following the user’s guide. Briefly, one unit of iTRAQ® reagent (an amount required to label 100 μg protein or phosphoprotein sample) was thawed and reconstituted in 24 μl of ACN (acetonitrile). Subsequently, a mixture of eight reconstituted samples was incubated for 2 h at room temperature, desalted and vacuum-dried. Finally, the sample was fractionated via high pH reverse-phase HPLC with an Agilent 300 Extend C18 column (5 μm particles, 4.6 × 250 mm). Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) over 80 min into 80 fractions, For the proteomic study, the peptides were combined into 14 fractions and dried by vacuum centrifuging. For the phosphoproteomic study, the peptides were combined into 4–6 fractions.
9
TMT Peptide Fractionation and Analysis
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TMT labeled peptides were solubilized in 500 μL solution containing 5% ACN/10 mM ammonium bicarbonate, pH 8.0 and separated by an Agilent 300 Extend C18 column (5 mm particles, 4.6 mm ID and 220 mm in length). An Agilent 1100 binary pump coupled with a photodiode array (PDA) detector (Thermo Scientific) was used to separate the peptides. A 40 minute linear gradient from 20% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8 (flow rate of 0.8 mL/min) separated the peptide mixtures into a total of 96 fractions (33 seconds). A total of 96 Fractions were consolidated into 12 samples in a checkerboard fashion, acidified with 20 μL of 20% formic acid and vacuum dried to completion. Each sample was re-dissolved in 5% FA/5% ACN, desalted via StageTips prior to LC-MS/MS analysis.
10
Neutrophil Proteome Profiling for Tuberculosis
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We pooled proteins from the NEUs from 15 individuals
into one sample,
namely, the H-NEU (healthy controls’ neutrophils) and P-NEU
(TB patients’ neutrophils) groups, respectively (combined with
different sexes and ages). Then equal proteins were mixed into a group
for three biological replicates namely H-NEU1, H-NEU2, H-NEU3 and
P-NEU1, P-NEU2, P-NEU3 groups, respectively. For digestion, the protein
solution was reduced with 5 mM dithiothreitol (Sigma) for 30 min at
56 °C and alkylated with 11 mM iodoacetamide (Sigma) for 15 min
at room temperature in darkness. The protein sample was then diluted
by adding 100 mM NH4HCO3 to urea concentration
less than 2M. Finally, trypsin (Promega) was added at 1:50 trypsin-to-protein
mass ratio for the first digestion overnight and 1:100 trypsin-to-protein
mass ratio for a second 4 h-digestion. These tryptic peptides were
fractionated into fractions by high-pH reverse-phase HPLC using Agilent
300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length).
Briefly, peptides were first separated with a gradient of 8% to 32%
acetonitrile (pH 9.0, Fisher Chemical) over 60 min into 60 fractions.
Then, the peptides were combined into three fractions and dried by
vacuum centrifuging.
into one sample,
namely, the H-NEU (healthy controls’ neutrophils) and P-NEU
(TB patients’ neutrophils) groups, respectively (combined with
different sexes and ages). Then equal proteins were mixed into a group
for three biological replicates namely H-NEU1, H-NEU2, H-NEU3 and
P-NEU1, P-NEU2, P-NEU3 groups, respectively. For digestion, the protein
solution was reduced with 5 mM dithiothreitol (Sigma) for 30 min at
56 °C and alkylated with 11 mM iodoacetamide (Sigma) for 15 min
at room temperature in darkness. The protein sample was then diluted
by adding 100 mM NH4HCO3 to urea concentration
less than 2M. Finally, trypsin (Promega) was added at 1:50 trypsin-to-protein
mass ratio for the first digestion overnight and 1:100 trypsin-to-protein
mass ratio for a second 4 h-digestion. These tryptic peptides were
fractionated into fractions by high-pH reverse-phase HPLC using Agilent
300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length).
Briefly, peptides were first separated with a gradient of 8% to 32%
acetonitrile (pH 9.0, Fisher Chemical) over 60 min into 60 fractions.
Then, the peptides were combined into three fractions and dried by
vacuum centrifuging.
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