Isobaric labeling of the peptides was accomplished with 6-plex TMT reagents (Thermo Scientific). Reagents, 0.8 mg, were dissolved in 40 μl acetonitrile (ACN), and 10 μl of the solution was added to 100 μg of peptides dissolved in 100 μl of 50 mM HEPES, pH 8.5. We found that the generation of unidentified and unwanted side reaction products – singly charged ions with m/z of 303.26, 317.26, and 331.29 – was prevented by using a 200 mM HEPES pH 8.5 buffer instead of the triethylammonium bicarbonate (TEAB) buffer recommended by the manufacturer. After 1 h at room temperature, the reaction was quenched by adding 8 μl of 5% hydroxylamine. Yeast peptides were labeled with all six reagents (126–131), human peptides were labeled with reagents 126, 127, and 128. Labeled peptides from yeast and human were separately mixed in the ratios described in the main manuscript and subjected to C18 SPE on Sep-Pak cartridges. After individual LC-MS2 analysis of both samples (
Hydroxylamine
It is a colorless, crystalline solid that is commonly used in organic synthesis, biochemistry, and as a reducing agent.
Hydroxylamine plays a key role in various biological processes, including the metabolism of certain amino acids and the production of nitric oxide.
Researchers can utilize PubCompare.ai to optimize their hydroxylamine-related studies by locating and comparing protocols from literature, preprints, and patents, ensuring reproducibility and accuracy.
This AI-driven platform enhances research by providing a comprehensive, SEO-optimized resource for hydroxylamine-focused investigations.
Most cited protocols related to «Hydroxylamine»
Isobaric labeling of the peptides was accomplished with 6-plex TMT reagents (Thermo Scientific). Reagents, 0.8 mg, were dissolved in 40 μl acetonitrile (ACN), and 10 μl of the solution was added to 100 μg of peptides dissolved in 100 μl of 50 mM HEPES, pH 8.5. We found that the generation of unidentified and unwanted side reaction products – singly charged ions with m/z of 303.26, 317.26, and 331.29 – was prevented by using a 200 mM HEPES pH 8.5 buffer instead of the triethylammonium bicarbonate (TEAB) buffer recommended by the manufacturer. After 1 h at room temperature, the reaction was quenched by adding 8 μl of 5% hydroxylamine. Yeast peptides were labeled with all six reagents (126–131), human peptides were labeled with reagents 126, 127, and 128. Labeled peptides from yeast and human were separately mixed in the ratios described in the main manuscript and subjected to C18 SPE on Sep-Pak cartridges. After individual LC-MS2 analysis of both samples (
model was prepared as previously.14 (link),16 (link) HeLa S3 cells
were grown in suspension to 1 × 106 cells/mL. Yeast
cells were grown to an OD of 1.0. Cells were lysed in 6 M guanidiumthiocyanate,
50 mM Hepes (pH 8.5, HCl). Protein content was measured using a BCA
assay (Thermo Scientific), disulfide bonds were reduced with dithiothreitol
(DTT), and cysteine residues were alkylated with iodoacetamide as
previously described.17 (link) Protein lysates
were cleaned with methanol–chloroform precipitation.18 (link) The samples were redissolved in 6 M guanidiumthiocyanate,
50 mM Hepes pH 8.5, and diluted to 1.5 M guanidium thiocyanate, 50
mM Hepes (pH 8.5). Both lysates were digested overnight with Lys-C
(Wako) in a 1/50 enzyme/protein w/w ratio. Following digestion, the
sample was acidified with TFA to a pH < 2 and subjected to C18 solid-phase extraction (SPE, Sep-Pak, Waters).
The
TMT reagents were dissolved in 40 μL of acetonitrile, and 10
μL of the solution was added to 100 μg of peptides dissolved
in 100 μL of 50 mM HEPES (pH 8.5). After incubating for 1 h
at room temperature (22 °C), the reaction was quenched by adding
8 μL of 5% w/v hydroxylamine. Following labeling, the sample
was combined in desired ratios. Yeast aliquots were mixed at 10:4:1:1:4:10,
and HeLa was mixed at 1:1:1:0:0:0 (Figure
to C18 solid-phase extraction.
Most recents protocols related to «Hydroxylamine»
Example 146
This compound was synthesized using CDI, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, and 6-(3-isoquinolyl)spiro[chromane-2,4′-piperidine] TFA salt. Analysis: LCMS m/z=474 (M+1); 1H NMR (400 MHz, CDCl3) δ: 9.30 (s, 1H), 8.00-7.95 (m, 2H), 7.92 (d, J=2.3 Hz, 1H), 7.88-7.82 (m, 2H), 7.68 (td, J=7.6, 1.1 Hz, 1H), 7.58-7.52 (m, 1H), 7.30 (s, 1H), 6.97 (d, J=8.5 Hz, 1H), 5.01-4.84 (m, 1H), 4.02-3.91 (m, 1H), 3.90-3.78 (m, 2H), 3.71-3.57 (m, 1H), 3.41-3.26 (m, 2H), 2.91 (t, J=6.8 Hz, 2H), 1.95-1.76 (m, 7H), 1.71-1.53 (m, 5H).
Example 171
This compound was synthesized using 5-(7-methylpyrazolo[1,5-a]pyridin-6-yl)spiro[3H-benzofuran-2,4′-piperidine] 2HCl and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine. Analysis: LCMS m/z=463 (M+1); 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.05 (d, J=2.3 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H), 7.28 (d, J=1.5 Hz, 1H), 7.21-7.11 (m, 2H), 6.87 (d, J=8.3 Hz, 1H), 6.68 (d, J=2.3 Hz, 1H), 4.76 (t, J=3.0 Hz, 1H), 4.02-3.93 (m, 1H), 3.56-3.43 (m, 3H), 3.43-3.34 (m, 2H), 3.10 (s, 2H), 2.65 (s, 3H), 1.89-1.44 (m, 10H).
Example 141
This compound was synthesized using O-(tetrahydro-2H-pyran-2-yl)hydroxylamine and 6-(5-methylimidazo[1,2-a]pyridin-6-yl)-N-tetrahydropyran-2-yloxy-spiro[chromane-2,4′-piperidine]-1′-carboxamide. 6-(5-Methylimidazo[1,2-a]pyridin-6-yl)-N-tetrahydropyran-2-yloxy-spiro[chromane-2,4′-piperidine]-1′-carboxamide Analysis: LCMS m/z=477 (M+1); 1H NMR (400 MHz, CDCl3) δ: 7.94 (d, J=10.0 Hz, 1H), 7.71 (s, 1H), 7.59-7.51 (m, 2H), 7.19 (d, J=9.3 Hz, 1H), 7.10-7.02 (m, 2H), 6.91 (d, J=8.3 Hz, 1H), 5.01-4.88 (m, 1H), 4.08-3.93 (m, 1H), 3.87 (br d, J=11.8 Hz, 2H), 3.68-3.58 (m, 1H), 3.33 (br t, J=12.9 Hz, 2H), 2.89-2.71 (m, 4H), 2.56 (s, 3H), 1.96-1.75 (m, 6H), 1.74-1.49 (m, 6H). 6-(5-Methylimidazo[1,2-a]pyridin-6-yl)spiro[chromane-2,4′-piperidine]-1′-carbohydroxamic acid Analysis: LCMS m/z=393 (M+1); 1H NMR (400 MHz, DMSO-d6) δ: 9.06 (s, 1H), 7.97 (s, 1H), 7.88 (s, 1H), 7.67 (d, J=1.3 Hz, 1H), 7.51 (d, J=9.0 Hz, 1H), 7.25-7.09 (m, 3H), 6.89 (d, J=8.3 Hz, 1H), 3.65 (br d, J=13.6 Hz, 2H), 3.15 (br t, J=10.7 Hz, 2H), 2.80 (br t, J=6.8 Hz, 2H), 2.55 (s, 3H), 1.83 (br t, J=6.8 Hz, 2H), 1.70 (br d, J=13.3 Hz, 2H), 1.61-1.48 (m, 2H).
Example 136
This compound was synthesized using 6-(8-chloro-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine] and O-(tetrahydro-2h-pyran-2-yl)hydroxylamine. 6-(8-Chloro-7-quinolyl)-N-tetrahydropyran-2-yloxy-spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxamide (0.125 g, 0.24 mmole) in DCM (5 mL) and TFA (2 mL) was stirred 2 h and concentrated. The product was purified by Gilson chromatography (5-45% ACN in water with 0.1% TFA). The pure fractions were concentrated, freebased and dried at 50° C. under vacuum. Analysis: LCMS m/z=426 (M+1); 1H NMR (400 MHz, DMSO-d6) δ: 9.15 (s, 1H), 9.06 (dd, J=4.1, 1.6 Hz, 1H), 8.49 (dd, J=8.3, 1.5 Hz, 1H), 8.09-7.99 (m, J=8.8 Hz, 2H), 7.67 (dd, J=8.3, 4.3 Hz, 1H), 7.62 (d, J=8.5 Hz, 1H), 7.40 (dd, J=8.4, 2.1 Hz, 1H), 7.31 (d, J=2.0 Hz, 1H), 7.00 (d, J=8.5 Hz, 1H), 4.95 (s, 2H), 3.51-3.35 (m, 4H), 1.93-1.80 (m, 4H).
Example 168
This compound was synthesized using 5-(8-methoxy-7-quinolyl)spiro[3H-benzofuran-2,4′-piperidine] 2HCl and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine. Analysis: LCMS m/z=490 (M+1); 1H NMR (400 MHz, DMSO-d6) δ: 9.73 (s, 1H), 8.94 (dd, J=4.3, 1.8 Hz, 1H), 8.38 (dd, J=8.3, 1.8 Hz, 1H), 7.75 (d, J=8.5 Hz, 1H), 7.57 (d, J=8.3 Hz, 1H), 7.54 (dd, J=8.3, 4.3 Hz, 1H), 7.47 (d, J=1.5 Hz, 1H), 7.38 (dd, J=8.3, 1.8 Hz, 1H), 6.88 (d, J=8.3 Hz, 1H), 4.76 (t, J=3.1 Hz, 1H), 4.02-3.95 (m, 1H), 3.91 (s, 3H), 3.53-3.44 (m, 3H), 3.43-3.34 (m, 2H), 3.12 (s, 2H), 1.89-1.43 (m, 10H).
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More about "Hydroxylamine"
This colorless, crystalline solid is widely used in organic synthesis, biochemistry, and as a reducing agent.
It plays a crucial role in various biological processes, including the metabolism of certain amino acids and the production of nitric oxide.
Researchers can leverage the power of PubCompare.ai to optimize their hydroxylamine-related studies.
This AI-driven platform enables users to locate and compare protocols from literature, preprints, and patents, ensuring reproducibility and accuracy.
By utilizing this comprehensive, SEO-optimized resource, researchers can enhance their investigations on hydroxylamine and related topics.
Hydroxylamine is closely associated with other important compounds and techniques, such as TMT reagent, Sep-Pak, TMT 10-plex reagents, Trypsin, Iodoacetamide, T-SOD assay kit, TMT10plex, and Sodium hydroxide.
These tools and reagents are often employed in conjunction with hydroxylamine-based research, providing researchers with a robust set of resources to explore the diverse applications and nuances of this remarkable chemical.
Whether you're studying the metabolic pathways involving hydroxylamine, investigating its role in nitric oxide production, or exploring its synthetic applications, PubCompare.ai can be a valuable asset in your research endeavors.
Unlock the full potential of your hydroxylamine-focused studies and take advantage of the insights and resources available through this innovative AI platform.