All LC–MS measurements were performed on a HPLC 1200 series (Agilent Technologies) connected to a 15T-SolariX XRTM Fourier Transform Ion-Cyclotron-Resonance Mass Spectrometer (FT-ICR-MS, Bruker Corp., Billerica, MA, USA). All proteins were digested online on a Nepenthesin-2 column (66 µL bed volume) in 0.4% Formic acid in water at 400 µL min−1 flow rate. The resulting peptides were trapped and desalted with the same buffer composition on a reversed-phase trap column (ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 2.1 mm × 5 mm, Waters, Milford, MA, USA). The desalted peptides were eluted and separated on an analytical reversed-phase column (ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 1 mm × 100 mm, Waters, Milford, MA, USA) with a 10–45% linear gradient of Solvent B (solvent A: 0.1% Formic acid in water, solvent B: 0.1% Formic acid, 2% water in Acetonitrile) at 40 µL min−1 flow rate. The column was connected directly to a FT-ICR-MS operated in positive data-dependent mode using collisional-induced dissociation. The raw data were processed in Data Analysis 5.0 (Bruker Corp., Billerica, MA, USA), and the peptides were identified using MASCOT (Matrix Science Ltd., UK) against a database containing DAPK2, 14-3-3γ, and Nepenthesin-2 (the only partial modifications allowed were Ser/Thr/Tyr phosphorylation, cysteine carbamidomethylation and single methionine oxidation).
Acquity uplc beh c18
Manufactured by Waters Corporation
Sourced in United States, Ireland, United Kingdom, Japan, Germany
The Acquity UPLC BEH C18 is a high-performance liquid chromatography (HPLC) column developed by Waters Corporation. It utilizes Waters' Bridged Ethylene Hybrid (BEH) particle technology and a C18 stationary phase, designed for the separation and analysis of a wide range of organic compounds.
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Lab products found in correlation
Acquity uplc, by Waters Corporation (13 mentions)
Acquity uplc system, by Waters Corporation (13 mentions)
Xevo tq s, by Waters Corporation (7 mentions)
Uv 2450 uv vis spectrophotometer, by Shimadzu (5 mentions)
Acquity uplc 1 class, by Waters Corporation (5 mentions)
Acquity uplc 1 class system, by Waters Corporation (5 mentions)
Avance 3 500 mhz spectrometer, by Bruker (4 mentions)
241 polarimeter, by PerkinElmer (4 mentions)
5 mm tci cryoprobe, by Bruker (4 mentions)
Avance 3 700 mhz spectrometer, by Bruker (4 mentions)
Bbfo plus smartprobe, by Bruker (4 mentions)
Xevo tqd triple quadruple instrument, by Waters Corporation (4 mentions)
Acquity uplc h class, by Waters Corporation (4 mentions)
Xevo g2 s qtof, by Waters Corporation (3 mentions)
Esi tof ms, by Bruker (3 mentions)
Acquity uplc beh c18 vanguard pre column, by Waters Corporation (3 mentions)
Masslynx software, by Waters Corporation (3 mentions)
Waters uplc system, by Waters Corporation (3 mentions)
Masslynx v4, by Waters Corporation (3 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (3 mentions)
281 protocols using acquity uplc beh c18
1
Protein Identification by FT-ICR-MS
2
UHPLC-MS/MS Quantification of Analytes
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Target analytes were separated and detected by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Chromatographic conditions were similar for EME and LLE samples. Gradient elution was carried out at 60 °C on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particles) analytical column with an Acquity UPLC BEH C18 (2.1 × 5 mm, 1.7 μm particles) pre-column on an Acquity UPLC I-Class instrument from Waters (Milford, MA, USA). Mobile phase A consisted of 5 mM NH4HCO2 (pH 10.1) and B consisted of MeOH. The flow rate was 0.5 mL/min and total run time 5.3 min. The gradient started with 5% B and continued to 30% B after 0.3 min, 50% at 2.7 min, 90% at 3.8 min and 98% at 4.8 min. The injection volume was 3 μL.
Analytes were detected using a Xevo TQ-S tandem mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray electrospray interface. Positive ionization (ESI+) was performed in multiple reaction monitoring (MRM). The capillary voltage was 1 kV and ion source temperature 120 °C. The desolvation gas (nitrogen) was heated to 650 °C and delivered with a flow rate of 1000 L/h. Cone gas flow was 150 L/h. MRM transitions of analytes included in the EME-UHPLC-MS/MS protocol are provided in TableS4 .
Analytes were detected using a Xevo TQ-S tandem mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray electrospray interface. Positive ionization (ESI+) was performed in multiple reaction monitoring (MRM). The capillary voltage was 1 kV and ion source temperature 120 °C. The desolvation gas (nitrogen) was heated to 650 °C and delivered with a flow rate of 1000 L/h. Cone gas flow was 150 L/h. MRM transitions of analytes included in the EME-UHPLC-MS/MS protocol are provided in Table
3
Purification of Crude Material via Preparative LC/MS
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Example 4104
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% B over 15 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
LC/MS (Injection 1 conditions) Rt=1.51 min, ESI m/z 745.1 (M+H).
LC/MS (Injection 2 conditions) Rt=1.35 min, ESI m/z 745.1 (M+H).
4
Purification of Crude Material via Preparative LC/MS
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Example 4004
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-65% B over 20 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
LCMS (Injection 1 conditions) Rt=1.51 min, ESI m/z 800.0 (M+H).
LCMS (Injection 2 conditions) Rt=1.47 min, ESI m/z 800.0 (M+H).
5
Purification of Desired Product via Preparative LC/MS
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Example 4060
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% B over 20 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. LCMS (Injection 1 conditions) Rt=1.50 min, ESI m/z 710.1 (M+H).
6
Purification of Crude Material via Preparative LC/MS
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Example 4102
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% B over 15 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
7
HPLC Purification and Characterization
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Example 4103
Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% B over 15 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. 729.1
LC/MS (Injection 1 conditions) Rt=1.51 min, ESI m/z 729.1 (M+H).
LC/MS (Injection 2 conditions) Rt=1.38 min, ESI m/z 729.1 (M+H).
8
Purification of Crude Material via Preparative LC/MS
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Example 4005
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 12-52% B over 23 minutes, then a 3-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
LCMS (Injection 1 conditions) Rt=1.50 min, ESI m/z 762.1 (M+H).
LCMS (Injection 2 conditions) Rt=1.40 min, ESI m/z 762.0 (M+H).
9
Purification of Organic Compound via LC/MS
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Example 4003
The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% B over 20 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Two analytical LC/MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
LCMS (Injection 1 conditions) Rt=1.42 min, ESI m/z 698.1 (M+H).
LCMS (Injection 2 conditions) Rt=1.38 min, ESI m/z 698.0 (M+H).
10
Enzymatic Reaction Analysis by UPLC-QTOF-MS
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Analyses were analyzed by UPLC-QTOF-MS (Waters Technologies, Milford, MA, United States) for the identification of reactions. Samples were separated on an ACQUITY UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters Technologies, United States) protected with an ACQUITY UPLC® BEH C18 guard column at 35°C, and the enzymatic products were eluted with a linear gradient condition given in Supplementary Table 2 . Then, 5 μl of sample was injected into the system. The conversion rates in percent were calculated from peak areas of products and substrates as analyzed by UPLC.
Time-of-flight MS detection was performed with a Xevo G2-S QTOF (Waters) system equipped with electrospray ionization (ESI) operating in negative ion mode. Full-scan monitoring range was performed in the range of m/z 50–1,500. The other operating parameters were as follows: the scanning time of 0.1 s; the detection time of 18 min; and the cone voltage of 40 V; and the high-energy ramp collision voltage was 20–50 V. Data acquisition and processing were performed using the MassLynx version 4.1 software (Waters Corp., Milford, MA, United States).
Time-of-flight MS detection was performed with a Xevo G2-S QTOF (Waters) system equipped with electrospray ionization (ESI) operating in negative ion mode. Full-scan monitoring range was performed in the range of m/z 50–1,500. The other operating parameters were as follows: the scanning time of 0.1 s; the detection time of 18 min; and the cone voltage of 40 V; and the high-energy ramp collision voltage was 20–50 V. Data acquisition and processing were performed using the MassLynx version 4.1 software (Waters Corp., Milford, MA, United States).
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