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Tandem Mass Spectrometry

Tandem Mass Spectrometry is a powerful analytical technique that combines two or more stages of mass spectrometry to provide detailed information about the structure and composition of complex molecules.
This method allows for the separation and identification of individual components within a sample, enabling researchers to gain deeper insights into a wide range of analytes, from small molecules to large biomolecules.
Tandem Mass Spectrometry offers unparalleled sensitivity, selectivity, and specifity, making it an indispensable tool in fields such as proteomics, metabolomics, and pharmacokinetics.
With its ability to provide high-resolution data and facilitate the discovery of novel compounds, Tandem Mass Spectrometry continues to be a cornerstone of modern analytical chemistry and bioscience reserach.

Most cited protocols related to «Tandem Mass Spectrometry»

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Publication 2010
Peptides Proteins SDS-PAGE Staphylococcal Protein A Tandem Mass Spectrometry Tissue Extracts Tissues Trypsin
An Escherichia coli K12 strain was grown in standard LB medium, harvested, washed in PBS, and lysed in BugBuster (Novagen Merck Chemicals, Schwalbach, Germany) according to the manufacturer's protocol. HeLa S3 cells were grown in standard RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% FBS. After being washed with PBS, cells were lysed in cold modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl, 1% N-octylglycoside, 0.1% sodium deoxycholate, complete protease inhibitor mixture (Roche)) and incubated for 15 min on ice. Lysates were cleared by centrifugation, and after precipitation with chloroform/methanol, proteins were resuspended in 6 m urea, 2 m thiourea, 10 mm HEPES, pH 8.0. Prior to in-solution digestion, 60-μg protein samples from HeLa S3 lysates were spiked with either 10 μg or 30 μg of E. coli K12 lysates based on protein amount (Bradford assay). Both batches were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with LysC (Wako Chemicals, GmbH, Neuss, Germany) for 4 h and then trypsin digested overnight (Promega, GmbH, Mannheim, Germany). Digestion was stopped by the addition of 2% trifluroacetic acid. Peptides were separated by isoelectric focusing into 24 fractions on a 3100 OFFGEL Fractionator (Agilent, GmbH, Böblingen, Germany) as described in Ref. 45 (link). Each fraction was purified with C18 StageTips (46 (link)) and analyzed via liquid chromatography combined with electrospray tandem mass spectrometry on an LTQ Orbitrap (Thermo Fisher) with lock mass calibration (47 (link)). All raw files were searched against the human and E. coli complete proteome sequences obtained from UniProt (version from January 2013) and a set of commonly observed contaminants. MS/MS spectra were filtered to contain at most eight peaks per 100 mass unit intervals. The initial MS mass tolerance was 20 ppm, and MS/MS fragment ions could deviate by up to 0.5 Da (48 (link)). For quantification, intensities can be determined alternatively as the full peak volume or as the intensity maximum over the retention time profile, and the latter method was used here as the default. Intensities of different isotopic peaks in an isotope pattern are always summed up for further analysis. MaxQuant offers a choice of the degree of uniqueness required in order for peptides to be included for quantification: “all peptides,” “only unique peptides,” and “unique plus razor peptides” (42 (link)). Here we chose the latter, because it is a good compromise between the two competing interests of using only peptides that undoubtedly belong to a protein and using as many peptide signals as possible. The distribution of peptide ions over fractions and samples is shown in supplemental Fig. S1.
Publication 2014
Acids Antibiotics, Antitubercular Biological Assay Buffers Cells Centrifugation Chloroform Cold Temperature Deoxycholic Acid, Monosodium Salt Digestion Dithiothreitol Edetic Acid Escherichia coli Escherichia coli K12 Glutamine HeLa Cells HEPES Homo sapiens Immune Tolerance Iodoacetamide Ions Isotopes Liquid Chromatography Methanol Peptides Promega Protease Inhibitors Proteins Proteome Radioimmunoprecipitation Assay Retention (Psychology) Sodium Chloride Staphylococcal Protein A Tandem Mass Spectrometry Thiourea Tromethamine Trypsin Urea
We describe here application of the UniDec approach to problems of increasing complexity: membrane protein AqpZ; small heat shock proteins HSP17.7, HSP16.5, and αB-crystallin; and lipoprotein Nanodiscs.
MS and IM-MS data of aquaporin Z (AqpZ) with bound 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) obtained at 100 V accelerating potential into a dedicated collision cell was analyzed using UniDec by limiting the mass range to between 95 and 105 kDa.27 (link) An example of how the algorithm performs without mass limitations is shown in Figure S-2. Data was smoothed in MassLynx 4.1 software (Waters Corp.) before analysis with Transform and MaxEnt, which used the same mass limitation.
Deconvolution of subunit exchange data from HSP17.7 was performed by limiting the allowed mass range to between 211 kDa and 222 kDa. Tandem MS spectra of the isolated +47 charge state of HSP16.5 24-mers were summed across multiple collision voltages to compile an aggregate spectrum.28 (link) Deconvolution was performed by limiting the charge state between 10 and 49 and manually defining the +47 charge state, which was necessary because only one charge state was isolated in the MS/MS experiment. Collision induced dissociation (CID) spectra of αB-crystallin were obtained similarly. Masses were limited to within 3000 Da of a wide range of potential oligomer complexes ranging from 1 to 74 subunits of a 20085 Da monomer. Charge was limited to between 5 and 84. In addition to the charge-smooth filter, a mass-smooth filter was applied to smooth the distribution of dimer units.
Nanodiscs with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC were analyzed with a linear drift cell Waters Synapt G1 ion mobility-mass spectrometer.29 (link) Data was deconvolved without a charge filter but by using a mass filter to smooth the distribution of lipids. Masses were limited to between 100 kDa and 175 kDa. Conversion from arrival time to collision cross section (CCS) was performed using the Mason-Schamp equation as described previously,27 (link),29 (link) using t0 values calibrated from alcohol dehydrogenase analyzed under the same instrumental conditions.
Publication 2015
Cells Crystallins Dehydrogenase, Alcohol Dimyristoylphosphatidylcholine Glycerylphosphorylcholine GPER protein, human Heat-Shock Proteins, Small Lipids Lipoproteins Phosphorylcholine plasma protein Z Protein Subunits Range of Motion, Articular Tandem Mass Spectrometry Tissue, Membrane Water Channel
All raw data were centroided and converted to 32-bit uncompressed mzXML file using Bruker Data Analysis. A script was developed to select all possible MS/MS spectra in each LC-MS/MS run that could correspond to a compound present in the sample. For each compound, we calculated the theoretical mass M from its chemical composition and searched for the M+H, M+2H, M+K, and M+Na adducts. Putative identifications included all MS/MS spectra whose precursor m/z had a ppm error <50 compared to the theoretical mass of each possible precursor m/z; all tandem MS/MS spectra with an MS1 precursor intensity of <1E4 were ignored. All candidate identifications were manually inspected and the most abundant representative spectrum for each compound was added to the corresponding library at the gold or bronze level based upon an expert evaluation of the spectrum quality. The best MS/MS spectrum per compound as added to the GNPS-Collections library without filtering or alteration from the mzXML files.
Publication 2016
cDNA Library chemical composition Gold Tandem Mass Spectrometry
A key step in placing statistically significant findings from chemometric analyses (as opposed to quantitative metabolomic analyses) into a biological context is to identify significantly altered compounds represented by certain spectral bins or certain clusters of spectral peaks. Once a user has identified lists of MS or NMR peaks that exhibit statistically significant changes, they may use one of several spectral comparison routines and spectral libraries to attempt to identify the compound(s) based on either lists of MS peaks (from MS or MS/MS data), GC-–MS peaks (from EI mass values and retention indices) or NMR peaks (from 1H, 13C or heteronuclear NMR spectra). These compound identification routines and spectral reference libraries were originally developed for the HMDB and for MetaboMiner (11 (link)). While not as comprehensive as some commercial libraries or commercial software, these freely available tools have been shown to be quite powerful in identifying many common compounds. Once compound information becomes available (via quantitative routes or via MetaboAnalyst's metabolite ID software), more insight can be obtained by which metabolic pathways are involved. Pathway mapping has been implemented in MetaboAnalyst using more than 70 pathway diagrams and metabolite libraries derived from the HMDB. Users simply type the names (or synonyms) of the metabolites identified and MetaboAnalyst provides the list of pathways in which these metabolites are found, along with hyperlinks to their pathway images. All results are linked to the HMDB where users can obtain more detailed information for each metabolite or pathway.
Publication 2009
Biopharmaceuticals Cortodoxone Gas Chromatography-Mass Spectrometry Nuclear Magnetic Resonance, Heteronuclear Retention (Psychology) Tandem Mass Spectrometry

Most recents protocols related to «Tandem Mass Spectrometry»

Example 8

Characterization of Absorption, Distribution, Metabolism, and Excretion of Oral [14C]Vorasidenib with Concomitant Intravenous Microdose Administration of [13C315N3]Vorasidenib in Humans

Metabolite profiling and identification of vorasidenib (AG-881) was performed in plasma, urine, and fecal samples collected from five healthy subjects after a single 50-mg (100 μCi) oral dose of [14C]AG-881 and concomitant intravenous microdose of [13C3 15N3]AG-881.

Plasma samples collected at selected time points from 0 through 336 hour postdose were pooled across subjects to generate 0—to 72 and 96-336-hour area under the concentration-time curve (AUC)-representative samples. Urine and feces samples were pooled by subject to generate individual urine and fecal pools. Plasma, urine, and feces samples were extracted, as appropriate, the extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectrometry (LC-MS and/or LC-MS/MS) analysis and by comparison of retention time with reference standards, when available.

Due to low radioactivity in samples, plasma metabolite profiling was performed by using accelerator mass spectrometry (AMS). In plasma, AG-881 was accounted for 66.24 and 29.47% of the total radioactivity in the pooled AUC0-72 h and AUC96-336 h plasma, respectively. The most abundant radioactive peak (P7; M458) represented 0.10 and 43.92% of total radioactivity for pooled AUC0-72 and AUC96-336 h plasma, respectively. All other radioactive peaks accounted for less than 6% of the total plasma radioactivity and were not identified.

The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), while no AG-881 was detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of dose in feces and for approximately 4% of dose in urine. M515, M460-1, M499, M516/M460-2, and M472/M476 were the most abundant metabolites in feces, and each accounted for approximately 2 to 5% of the radioactive dose, while M266 was the most abundant metabolite identified in urine and accounted for a mean of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for <1% of the dose.

Overall, the data presented indicate [14C]AG-881 underwent moderate metabolism after a single oral dose of 50-mg (100 μCi) and was eliminated in humans via a combination of metabolism and excretion of unchanged parent. AG-881 metabolism involved the oxidation and conjugation with glutathione (GSH) by displacement of the chlorine at the chloropyridine moiety. Subsequent biotransformation of GSH intermediates resulted in elimination of both glutamic acid and glycine to form the cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further converted by a series of biotransformation reactions such as oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation and N-dealkylation resulting in the formation multiple metabolites.

A summary of the metabolites observed is included in Table 2

TABLE 2
Retention
ComponentTimeMatrix
designation(Minutes)[M + H]+Type of BiotransformationPlasmaUrineFeces
Unidentified 17.00UnknownX
M2667.67a267N-dealkylationX
Unidentified 2UnknownX
Unidentified 3UnknownX
Unidentified 4UnknownX
Unidentified 5UnknownX
M51519.79b516OxidationX
M460-120.76b461OxidationX
M49921.22b500Dechloro-glutathioneXX
conjugation + hydrolysis
M51621.89b517Oxidative-deaminationX
M460-221.98b461OxidationX
M47222.76b473S-dealkylation + S-X
acetylation + reduction
M47622.76b477OxidationX
Unidentified 6UnknownX
M47423.63b475OxidationX
Unidentified 7UnknownX
M43025.88b431AG-881-oxidationX
M42630.62b427S-dealkylation + methylationX
M45831.03c459AG-69460X*
AG-88139.41b415AG-881XX
M42847.40b429S-dealkylation + oxidationX
Table 3 contains a summary of protonated molecular ions and characteristic product ions for AG-881 and identified metabolites

TABLE 3
RetentionCharacteristic
MetaboliteTimeProposed MetaboliteProduct Ions
designation(Minutes)[M + H]+Identification(m/z)Matrix
M266 7.88a267[Figure (not displayed)]
188, 187Urine
M51519.79b516[Figure (not displayed)]
429, 260, 164, 153Feces
M460-120.76b461[Figure (not displayed)]
379, 260, 164Feces
M49921.22b500[Figure (not displayed)]
437, 413, 260, 164, 137Urine Feces
M51621.89b517[Figure (not displayed)]
427, 260, 164, 153Feces
M460-221.98b461[Figure (not displayed)]
369, 260, 164, 139, 121, 93Feces
M47222.76b473[Figure (not displayed)]
429, 260, 179, 164, 153Feces
M47622.76b477[Figure (not displayed)]
395, 260, 164, 139, 119Feces
M47423.63b475[Figure (not displayed)]
260, 164, 68Feces
M43025.88b431[Figure (not displayed)]
260, 164, 155, 68Feces
M42630.62b427[Figure (not displayed)]
260, 164, 151Feces
M45831.03b459[Figure (not displayed)]
380, 311, 260, 183, 164, 130Plasma Fecesd
AG-88139.41b415[Figure (not displayed)]
319, 277, 260, 240, 164, 139, 119, 68Plasma Fecesd
M42847.40b429[Figure (not displayed)]
260, 164, 153Feces
Notes
aRetention time from analysis of a urine sample
bRetention time from analysis of a feces sample
cRetention time from analysis of a plasma sample
dM458 was only detected in feces by mass spectrometry, not by radioprofiling.
The proposed (theoretical) biotransformation pathways leading to the observed metabolites are shown in FIG. 1.

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Patent 2024
Acetylation AG 30 Biotransformation Chlorine Dealkylation Deamination Elements, Radioactive Feces Glutamic Acid Glutathione Glycine Healthy Volunteers High-Performance Liquid Chromatographies Homo sapiens Hydrolysis Intravenous Infusion Ions Liquid Chromatography Mass Spectrometry Metabolism Methylation Parent Plasma Radioactivity Retention (Psychology) Tandem Mass Spectrometry Urinalysis Urine vorasidenib

Example 2

[Figure (not displayed)]

N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(5-ethyl-2-morpholino-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate B) (200 mg, 352 μmol) was suspended in DMF (5 mL). Perfluorophenyl 3-hydroxypicolinate (Intermediate CT) (215 mg, 703 μmol) and Et3N (97.0 μL, 703 μmol) were added and the RM was stirred at 70° C. for 3 hours. The RM was concentrated under reduced pressure. The crude product was first purified by column chromatography (Silica gel column: Silica 12 g, eluent DCM:MeOH 100:0 to 90:10). Then a second purification by reverse phase preparative HPLC (RP-HPLC acidic 9: 40 to 50% B in 2 min, 50 to 55% B in 10 min) afforded the title compound.

LC-MS: Rt=0.98 min; MS m/z [M+H]+ 690.6/692.6, m/z [M−H] 688.4/690.3; UPLC-MS 1

LC-MS: Rt=4.84 min; MS m/z [M+H]+ 690.2/692.2 m/z [M−H] 688.3/690.3; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, br, 1H), 10.34 (s, br, 1H), 8.05 (m, 2H), 7.96 (d, J=2.1 Hz, 1H), 7.72 (dd, J=2.1 Hz, 8.7 Hz, 1H), 7.28 (m, 2H), 5.21 (s, 2H), 4.53 (m, 1H), 3.66 (m, 4H), 3.46 (m, 3H), 3.38 (m, 4H), 3.20 (m, 1H), 2.92 (m, 3H), 2.76 (m, 1H), 2.58 (m, 1H), 1.16 (t, J=7.5 Hz, 3H)

Example 24

[Figure (not displayed)]

To the stirred solution of N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-2-(4-methoxycyclohex-1-en-1-yl)-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate Y) (300 mg, 504 μmol), 4-chloro-3-hydroxypicolinic acid (140 mg, 807 μmol), HOBt (136 mg, 1.01 mmol) and EDC.HCl (193 mg, 1.01 mmol) in DCM (20 mL) was added pyridine (122 μL, 1.51 mmol) at 0° C. The RM was stirred at RT for 16 hours. The RM was quenched with NaHCO3 and extracted with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (Silica gel column: Silica 4 g, eluent DCM:MeOH 100:0 to 98:2). The residue was purified by preparative chiral HPLC (instrument: Agilent 1200 series, with single quad mass spectrometer; column: LUX CELLULOSE-4, 250 mm×21.1 mm, 5.0 μm; eluent: A=hexane, B=0.1% HCOOH in EtOH; flow rate: 15 mL/min; detection: 210 nm; injection volume: 0.9 mL; gradient: isocratic: 50(A):50(B)).

Example 24a: The product containing fractions were concentrated at 40° C. and washed with n-pentane (5×10 mL), decanted and dried to give the title compound as an off-white solid—first eluting stereoisomer.

Chiral HPLC (C-HPLC 2): Rt=10.764 min

LC-MS: Rt=1.08 min; MS m/z [M+H]+ 750.5/752.5, m/z [M−H] 748.4/750.4; UPLC-MS 1

LC-MS: Rt=5.29 min; MS m/z [M+H]+ 750.2/752.2, m/z [M−H] 748.2/750.2; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, br, 2H), 8.56 (d, J=8.1 Hz, 1H), 7.98 (d, J=5.6 Hz, 1H), 7.94 (d, J=8.1 Hz, 1H), 7.50 (d, J=5.1 Hz, 1H), 6.72 (m, 1H), 5.34 (s, 2H), 4.53 (m, 1H), 3.52 (m, 4H), 3.28 (m, 4H), 2.98 (m, 3H), 2.80 (m, 1H), 2.63 (m, 1H), 2.55 (m, 1H), 2.46 (m, 1H), 2.16 (m, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.17 (t, J=7.3 Hz, 3H)

Example 24b: The product containing fractions were concentrated at 40° C. and washed with n-pentane (5×10 mL), decanted and dried to give the title compound as an off-white solid—second eluting stereoisomer.

Chiral HPLC (C-HPLC 2): Rt=18.800 min

LC-MS: Rt=1.08 min; MS m/z [M+H]+ 750.1/752.1, m/z [M−H] 748.2/750.2; UPLC-MS 1

LC-MS: Rt=5.30 min; MS m/z [M+H]+ 750.1/752.1, m/z [M−H] 748.2/750.2; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, br, 1H), 10.55 (s, br, 1H), 8.56 (d, J=8.2 Hz, 1H), 8.06 (d, J=5.3 Hz, 1H), 7.92 (d, J=8.2 Hz, 1H), 7.55 (d, J=5.3 Hz, 1H), 6.72 (m, 1H), 5.35 (s, 2H), 4.54 (m, 1H), 3.54 (m, 4H), 3.28 (m, 3H), 3.25 (m, 1H), 2.99 (m, 3H), 2.81 (m, 1H), 2.62 (m, 1H), 2.41 (m, 2H), 2.16 (m, 2H), 1.96 (m, 1H), 1.66 (m, 1H), 1.18 (t, J=7.3 Hz, 3H)

Example 25

[Figure (not displayed)]

N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-2-(4-methoxycyclohex-1-en-1-yl)-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide.HCl (Intermediate Y) (120 mg, 190 μmol) and DIPEA (166 μL, 950 μmol) were dissolved in DCM (5 mL) and then 3-hydroxypicolinoyl chloride (Intermediate CV) (59.9 mg, 380 μmol) was added at 0° C. and stirred for 2 hours. 3-hydroxypicolinoyl chloride (Intermediate CV) (59.9 mg, 380 μmol) was added again and the reaction was continued under stirring for 12 hours. The RM was diluted with DCM and washed with water and aq NaHCO3 (2×20 mL), washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude product was combined with another experiment and purified by column chromatography (Silica gel column: Silica 4 g, eluent DCM:MeOH 100:0 to 99:1) then further purified by reverse phase preparative HPLC (RP-HPLC acidic 10: 40 to 50% B in 2 min, 50 to 60% B in 8 min) to give the title compound as an off-white solid.

The racemate was purified by preparative chiral HPLC (instrument: Agilent 1200 series, with single quad mass spectrometer; column: CELLULOSE-4, 250 mm×21.2 mm; eluent: A=hexane, B=0.1% HCOOH in MeOH:EtOH 1:1; flow rate: 20 mL/min; detection: 210 nm; injection volume: 0.9 mL; gradient: isocratic 60(A):40(B)).

Example 25a: First eluting stereoisomer, off-white solid.

Chiral HPLC (C-HPLC 1): Rt=10.070 min

LC-MS: Rt=0.98 min; MS m/z [M+H]+ 716.5/718.6, m/z [M−H] 714.3/716.3; UPLC-MS 1

LC-MS: Rt=4.76 min; MS m/z [M+H]+ 716.2/718.2, m/z [M−H] 714.2/716.2; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, br, 2H), 8.56 (d, J=8.5 Hz, 1H), 8.05 (m, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.28 (m, 2H), 6.72 (m, 1H), 5.30 (s, 2H), 4.54 (m, 1H), 3.47 (m, 4H), 3.27 (s, 3H), 3.21 (m, 1H), 2.96 (m, 3H), 2.79 (m, 1H), 2.59 (m, 3H), 2.43 (m, 1H), 2.14 (m, 1H), 1.95 (m, 1H), 1.67 (m, 1H), 1.17 (t, J=7.2 Hz, 3H)

Example 25b: Second eluting stereoisomer, off-white solid.

Chiral HPLC (C-HPLC 1): Rt=16.023 min

LC-MS: Rt=0.96 min; MS m/z [M+H]+ 716.3/718.3, m/z [M−H] 714.3/716.3; UPLC-MS 1

LC-MS: Rt=4.77 min; MS m/z [M+H]+ 716.2/718.2, m/z [M−H] 714.2/716.2; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, br, 2H), 8.56 (d, J=8.0 Hz, 1H), 8.06 (m, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.28 (m, 2H), 6.72 (m, 1H), 5.32 (s, 2H), 4.54 (m, 1H), 3.46 (m, 4H), 3.27 (s, 3H), 3.20 (m, 1H), 2.96 (m, 3H), 2.79 (m, 1H), 2.59 (m, 3H), 2.41 (m, 1H), 2.14 (m, 1H), 1.95 (m, 1H), 1.68 (m, 1H), 1.17 (t, J=7.1 Hz, 3H)

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Patent 2024
1-hydroxybenzotriazole 1H NMR acetamide Acids Bicarbonate, Sodium Bicyclo Compounds brine Cellulose Chlorides Chromatography DIPEA Ethanol H 718 Hexanes High-Performance Liquid Chromatographies Morpholinos pentane Piperazine Pressure pyridine Silica Gel Silicon Dioxide Stereoisomers Sulfoxide, Dimethyl Tandem Mass Spectrometry

Example 18

[Figure (not displayed)]

To the stirred solution of 3-hydroxypicolinic acid (166 mg, 1.19 mmol), EDC.HCl (228 mg, 1.19 mmol), HOBt (161 mg, 1.19 mmol) in DMF (3 mL) was added N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-7-oxo-6-(piperazin-1-yl)-2-(pyrrolidin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate Q) (330 mg, 596 μmol) and DIPEA (624 μL, 3.57 mmol) and the RM was at RT for 16 hours. The RM was concentrated under reduced pressure and water was added. The resultant brown solid was filtered off and dried under vacuum. The crude product was purified by reverse phase preparative HPLC (RP-HPLC acidic 4: 35 to 40% B in 2 min, 40 to 45% B in 10 min) to give the title compound.

LC-MS: Rt=0.94 min; MS m/z [M+H]+ 675.3/677.3, m/z [M−H] 673.3/675.3; UPLC-MS 1

LC-MS: Rt=4.68 min; MS m/z [M+H]+ 675.2/677.2, m/z [M−H] 673.2/675.2; UPLC-MS 2

1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, br, 1H), 10.38 (s, br, 1H), 8.55 (d, J=8.4 Hz, 1H), 8.06 (m, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.28 (m, 2H), 5.25 (s, 2H), 4.53 (m, 1H), 3.46 (m, 3H), 3.35 (m, 4H), 3.19 (m, 1H), 2.91 (m, 3H), 2.75 (m, 1H), 2.57 (m, 1H), 1.89 (m, 4H), 1.15 (t, 3H)

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Patent 2024
1-hydroxybenzotriazole 1H NMR acetamide Acids DIPEA High-Performance Liquid Chromatographies Piperazine Pressure Sulfoxide, Dimethyl Tandem Mass Spectrometry Vacuum
Not available on PMC !

Example 8

The SFME sample processing can be done in fused silica tubing of smaller diameter which are commonly used as liquid line in liquid chromatography system (e.g., tubing having an inner diameter of 500 μm or less). The extraction can be induced by applying a push and pull force on one side of the tubing. The extract can be either directly analyzed by nanoESI or stored for further operations.

FIG. 10 show analysis of 50 ng/mL amitriptyline in bovine whole blood. MS/MS spectrum of the molecular ion was collected. The blood sample was first 10× diluted using H2O as a reduction of viscosity. For extraction, 5 μL of the diluted sample was processed in a fused silica tubing (i.d. 500 μm) using methods of the invention. The extract was then infused into a nanoESI emitter and analyzed by nanoESI.

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Patent 2024
Amitriptyline BLOOD Cattle Liquid Chromatography Silicon Dioxide Tandem Mass Spectrometry Viscosity
Not available on PMC !

Example 10

The objective of this study was to develop an acute model of homocystinuria in nonhuman primates. Male cynomolgus monkeys of approximately 2-5 years of age (average weight of 3.4 kg) were fasted overnight and orally administered a methionine load at 100 or 300 mg/kg, and plasma was collected at 0-, 0.5-, 1-, 2-, 4-, 6-, and 24-hours post-dose for methionine and total homocysteine measurements by LC-MS/MS.

Oral administration of methionine (100 or 300 mg/kg) resulted in a dose-dependent increase in plasma methionine levels, with peak concentration recorded at 30 minutes and 1 hour post dose for 100 mg/kg and 300 mg/kg, respectively (FIG. 16A). Plasma methionine concentrations gradually decreased over time and reached pre-dose levels by 24 hours. The oral methionine load also resulted in a significant elevation in total plasma homocysteine by 30 minutes post dose, but no statistically significant difference between groups was noted (FIG. 16B). By 24 hours, total homocysteine levels had returned to baseline values for both groups. In conclusion, this study indicates that oral administration of a methionine load to nonhuman primates leads to acute homocystinuria.

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Patent 2024
Administration, Oral Homocysteine Homocystinuria Macaca fascicularis Males Methionine Plasma Primates Tandem Mass Spectrometry

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Trypsin is a serine protease enzyme that is commonly used in cell culture and molecular biology applications. It functions by cleaving peptide bonds at the carboxyl side of arginine and lysine residues, which facilitates the dissociation of adherent cells from cell culture surfaces and the digestion of proteins.
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Mascot is a versatile lab equipment designed for efficient sample preparation and analysis. It features a compact and durable construction, enabling reliable performance in various laboratory settings.
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Proteome Discoverer is a software solution for the analysis of mass spectrometry-based proteomic data. It provides a comprehensive platform for the identification, quantification, and characterization of proteins from complex biological samples.
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The Q Exactive is a high-resolution mass spectrometer designed for accurate and sensitive analysis of a wide range of samples. It features a quadrupole mass filter and an Orbitrap mass analyzer, providing high-resolution, accurate mass measurements for qualitative and quantitative applications.
Sourced in United States, Canada, Germany, Singapore, United Kingdom
Analyst 1.6.3 software is a data processing and analysis tool developed by AB Sciex for use with their mass spectrometry instruments. The software provides functionalities for data acquisition, processing, and visualization. It serves as a core component in the operation and analysis of mass spectrometry experiments.
Sourced in United States, United Kingdom, Germany
Proteome Discoverer 1.4 is a software application designed for the analysis and identification of proteins in mass spectrometry data. It provides a platform for processing, analyzing, and interpreting proteomics data.
Sourced in United States, Germany, Denmark, Japan
The EASY-nLC 1000 is a high-performance liquid chromatography (nanoLC) system designed for sensitive and reproducible separation of complex peptide mixtures. It offers precise solvent delivery, robust performance, and compatibility with a range of detectors, making it a versatile tool for proteomics research and analysis.
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The Q Exactive Plus is a high-resolution, accurate-mass Orbitrap mass spectrometer designed for a wide range of applications, including proteomics, metabolomics, and small molecule analysis. It features high-performance mass analysis, with a mass resolution up to 280,000 FWHM at m/z 200 and mass accuracy of less than 1 ppm.
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The Thermo Scientific™ Ultimate 3000 is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a modular design, allowing for customization to meet specific laboratory needs.

More about "Tandem Mass Spectrometry"

Tandem mass spectrometry (MS/MS or MS2) is a powerful analytical technique that combines multiple stages of mass spectrometry to provide in-depth information about the structure and composition of complex molecules.
This method allows for the separation, identification, and characterization of individual components within a sample, enabling researchers to gain deeper insights into a wide range of analytes, from small molecules to large biomolecules.
Tandem mass spectrometry offers unparalleled sensitivity, selectivity, and specificity, making it an indispensable tool in fields such as proteomics, metabolomics, and pharmacokinetics.
The technique typically involves an initial stage of mass spectrometry to isolate and select precursor ions, followed by one or more subsequent stages of fragmentation and mass analysis to generate detailed structural information.
Q Exactive mass spectrometers, combined with powerful software like Proteome Discoverer and Analyst 1.6.3, are commonly used in tandem mass spectrometry workflows.
These instruments provide high-resolution data and facilitate the discovery of novel compounds, making them essential for modern analytical chemistry and bioscience research.
Trypsin, a commonly used enzyme in proteomics studies, is often employed in tandem mass spectrometry experiments to generate peptide fragments for analysis.
The Mascot search engine is a widely used tool for the identification of proteins from tandem mass spectrometry data.
With its ability to provide detailed information about complex samples, tandem mass spectrometry continues to be a cornerstone of modern analytical techniques.
The EASY-nLC 1000 and Q Exactive Plus systems further enhance the capabilities of this powerful method, enabling researchers to push the boundaries of their investigations and make groundbreaking discoveries.