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Asp n

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Asp-N is a lab equipment product manufactured by Promega. It is a proteolytic enzyme that cleaves peptide bonds specifically on the C-terminal side of aspartic acid residues in proteins.

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Lab products found in correlation

Trypsin, by Promega (13 mentions) Chymotrypsin, by Promega (11 mentions) Glu c, by Promega (10 mentions) Lys c, by Promega (5 mentions) Urea, by Merck Group (4 mentions) Pngase f, by Promega (4 mentions) Sodium chloride (nacl), by Merck Group (4 mentions) Acetonitrile, by Thermo Fisher Scientific (4 mentions) Methylamine, by Merck Group (3 mentions) Cacl2, by Merck Group (3 mentions) Sequencing grade modified trypsin, by Promega (3 mentions) Iodoacetamide, by Merck Group (3 mentions) Pepsin, by Promega (3 mentions) Arg c, by Promega (3 mentions) Trypsin lys c mix, by Promega (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Hepes, by Merck Group (2 mentions) 2 iodoacetamide, by Thermo Fisher Scientific (2 mentions) Dextran sulfate sodium (dss), by Thermo Fisher Scientific (2 mentions) Alpha lytic protease, by New England Biolabs (2 mentions)

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Asp-N-protease (2 citations)

34 protocols using asp n

1

Asp-N Digestion of Flagellin

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For Asp-N digestion, 3 µg of flagellin was reconstituted in 17 µL of 2 M urea containing 50 mM ammonium bicarbonate, and digested by 0.08 µg of Asp-N (Promega) at 37 °C for overnight. The digestion was stopped by adding 1/20 volume of 20% TFA, and digested peptides were desalted using a GL-Tip SDB (GL Science) according to the manufacturer’s instructions. After vacuum concentration, the samples were dissolved in 20 µL of 2% acetonitrile (containing 0.1% TFA) and 1.5-µL aliquots were analyzed by nano-LC-MS/MS.
Matsui S., Noda S., Kuwata K., Nomoto M., Tada Y., Shinohara H, & Matsubayashi Y. (2024). Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin. Nature Communications, 15, 3762.
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2

Purification and Analysis of MHC Proteins

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All reagents were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA) unless noted otherwise. Glu-C, Asp-N and trypsin endoproteinases were purchased from Promega (Madison, WI, USA). Standard MHC protein purified from porcine heart was purchased from Sigma-Aldrich (St Louis, MO, USA). All solutions were prepared with HPLC grade water (Fisher Scientific, Fair Lawn, NJ, USA).
Jin Y., Wei L., Cai W., Lin Z., Wu Z., Peng Y., Kohmoto T., Moss R.L, & Ge Y. (2017). Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-down Mass Spectrometry. Analytical chemistry, 89(9), 4922-4930.
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3

Mass Spectrometry Sample Preparation

Cited 1 time
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DSS, tris(2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), and 2-Iodoacetamide (IAA) were purchased from Pierce Biotechnology (Thermo Scientific). Dimethylsulfoxide (DMSO), HEPES, NaCl, urea, CaCl2, and methylamine were purchased from Sigma-Aldrich. Acetonitrile (ACN), formic acid (FA), acetone, and ammonium bicarbonate were purchased from J.T. Baker. Trypsin and Asp-N (gold mass spectrometry grade) were purchased from Promega. DOPA2 was synthesized as previously described (Wang et al.2022 (link)).
Wang J.H., Gong Z., Dong X., Liu S.Q., Tang Y.L., Lei X., Tang C, & Dong M.Q. (2022). Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes. Biophysics Reports, 8(5-6), 239-252.
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4

SARS-CoV-2 Spike Protein Production

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All
chemicals were obtained from Sigma-Aldrich
(St. Louis, MO) except where otherwise indicated. Endoproteases including
trypsin, chymotrypsin, Asp-N, and Lys-C were purchased from Promega
(Madison, WI). Recombinant SARS-CoV-2 prefusion stabilized spike protein
was provided by Dr. Jason McLellan at University of Texas at Austin.
The protein was expressed in human FreeStyle293F cells.12 (link) Recombinant SARS-CoV-2 spike protein expressed
in baculovirus insect cells (S1 + S2 ECD, Cat. no. 40589-V08B1), SARS-CoV-2
spike protein S1 subunit (Cat. no. 40591-V08H), and MERS-CoV spike
protein S1 subunit, both expressed in human-derived HEK cells (Cat.
no. 40069-V08H) were purchased from Sino Biological (Wayne, PA). SARS-CoV-1
spike protein (Cat. no. NR-686) prepared in insect cells was obtained
from BEI Resources (Manassas, VA).
Wang D., Baudys J., Bundy J.L., Solano M., Keppel T, & Barr J.R. (2020). Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry. Analytical Chemistry, 92(21), 14730-14739.
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5

Expression of Epitope-Tagged DDHD1 Mutant

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Oligonucleotides for the expression of the epitope-tagged DDHD1 mutant in HEK293 and PNAC1 cells described below were synthesized by Invitrogen. Sequencing-grade modified trypsin and AspN were purchased from Promega. High-purity MS grade 2, 5-dihydroxybenzoic acid (DHB) was from Shimadzu. α-Cyano-4-hydroxycinnamic acid (α-CHCA) was from Sigma. Phos-tag acrylamide was from Wako. The mixture of protease inhibitors containing 4-(2-aminoethyl) benzenesulfonyl fluoride, aprotinin, bestatin, E-64, leupeptin, and pepstatin A was from Calbiochem. Olomoucine was from Sigma. CHIR99021 was from Wako. Anti-mouse IgG antibodies conjugated with horseradish peroxidase were from Dako. All other reagents were from Wako, unless otherwise mentioned. All other compounds were of reagent grade or higher. The expression plasmid for paxillin α (35 (link)) was a generous gift of Dr M. Nishita (Fukushima Medical University).
Matsumoto N., Nemoto-Sasaki Y., Oka S., Arai S., Wada I, & Yamashita A. (2021). Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization. The Journal of Biological Chemistry, 297(1), 100851.
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6

Quantitative SEPT2 Proteomic Profiling

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Proteins were separated by SDS-PAGE. After aligning of anti-SEPT2 western blot, gel bands corresponding to full length SEPT2 and C-terminal SEPT2 fragment were excised and digested using trypsin then propionic anhydride labeling followed by digestion by either trypsin or Asp-N (Promega). Extracted peptides were analyzed by LC-MS/MS (EasyLC 1200 coupled to Fusion Lumos, Thermo Scientific), separated by reversed phase with a gradient increasing from 1% B to 40% B in 40 min. MS data were queried against Uniprots Human database (March 2016) using no enzymatic (Asp-N experiment) or semi-tryptic constraints using Proteome Discoverer ver1.4 (Thermo Fisher)/Mascot 2.4 (Matrix Science). Oxidation of methionine was allowed. For samples treated with propionic anhydride, propionylation of N-termini and lysine were allowed as variable modifications. Matched peptides were filtered using < 5% FDR (Percolator 2) and mass accuracy < 5 ppm in addition to manual validation of tandem spectra. Extracted areas of Septin-2 peptides obtained by the three different digestion strategies for intact SEPT2 and C-terminal SEPT2 were summed per residue and plotted as a function of SEPT2 using PepEx 3, after normalization to the signal of the most C-terminal residues.
Li H., Saucedo-Cuevas L., Yuan L., Ross D., Johansen A., Sands D., Stanley V., Guemez-Gamboa A., Gregor A., Evans T., Chen S., Tan L., Molina H., Sheets N., Shiryaev S.A., Terskikh A.V., Gladfelter A.S., Shresta S., Xu Z, & Gleeson J.G. (2019). Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors. Neuron, 101(6), 1089-1098.e4.
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7

Gel Electrophoresis and Protein Digestion

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4–15% Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well, 15 μl (Bio-Rad); SimplyBlue™ SafeStain (Invitrogen); dithiothreitol (Sigma); iodoacetamide (Sigma); alpha lytic protease (New England BioLabs); chymotrypsin (Athens Research and Technology); AspN (Promega); Glu-C (Promega); trypsin (Promega); endoglycosidase H (Promega); PNGaseF (Promega); 18O water (Cambridge Isotope Laboratories).
Chmielewski D., Wilson E.A., Pintilie G., Zhao P., Chen M., Schmid M.F., Simmons G., Wells L., Jin J., Singharoy A, & Chiu W. (2023). Integrated analyses reveal a hinge glycan regulates coronavirus spike tilting and virus infectivity. Research Square.
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8

Glycoprotein Enrichment and Analysis

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For the label-free
samples, glycoproteins were reduced with 5 mM TCEP for 30 min at 37
°C, then alkylated with 15 mM iodoacetamide in the dark at room
temperature for 30 min. Trypsin (Promega, Madison, WI) was added to
digest protein at 37 °C overnight with a ratio of enzyme to protein
of 1:30. The TMT labeled glycoproteins were divided into two equal
fractions and digested with Trypsin or Asp-N (Promega, Madison, WI)
at 37 °C overnight. Glycopeptides were deglycosylated using PNGase
F (New England Biolabs, Ipswich, MA) at 37 °C for 16 h and dried
using a SpeedVac concentrator (Thermo Savant, Milford, MA). The samples
were desalted using C18 ZipTips (Millipore, Billerica,
MA) before LC-MS/MS analysis.
Nie S., Lo A., Wu J., Zhu J., Tan Z., Simeone D.M., Anderson M.A., Shedden K.A., Ruffin M.T, & Lubman D.M. (2014). Glycoprotein Biomarker Panel for Pancreatic Cancer Discovered by Quantitative Proteomics Analysis. Journal of Proteome Research, 13(4), 1873-1884.
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9

SARS-CoV-2 Spike Protein Characterization

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All chemicals were obtained from Sigma–Aldrich (St. Louis, MO) unless indicated otherwise. Endoproteases used in this study, including trypsin (Cat#XV528X, Mass Spectrometry Grade), chymotrypsin (Cat#V1061, Sequencing Grade), Lys-C (Cat#VA1170, Mass Spectrometry Grade), and Asp-N (Cat#V162A, Sequencing Grade), were obtained from Promega (Madison, WI), and alpha-lytic protease (Cat#P8113S, Proteomics Grade) was purchased from New England BioLabs (Ipswich, MA). The recombinant poly-histidine tagged, trimeric ectodomains of the SARS-CoV-2 S variants (D614G, Alpha, Beta, Gamma, and Delta) expressed in HEK293 cells were purchased from R&D Systems (Minneapolis, MN). The sequences of the two S proteins included a mutated furin cleavage site and K986P, V987P (2P) substitutions.
Wang D., Baudys J., Osman S.H, & Barr J.R. (2023). Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2. Analytical and Bioanalytical Chemistry, 415(19), 4779-4793.
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10

Proteome Analysis Reagents and Enzymes

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The following reagents were purchased from Sigma-Aldrich (Shanghai Titan Scientific Co., Ltd.): dithiothreitol, iodoacetamide, ammonium sulfate, urea, and acetone. MS-grade formic acid (FA) and acetonitrile were from Thermo Fisher Scientific. ProteaseMAX Surfactant, PNGaseF, pepsin, trypsin, chymotrypsin, LysC, GluC, and AspN were from Promega.
Fan H., Wang B., Shi L., Pan N., Yan W., Xu J., Gong L., Li L., Liu Y., Du C., Cui J., Zhu G., Deng S., Sui W., Xu Y., Yi S., Hao M., Zou D., Chen X., Qiu L, & An G. (2024). Monitoring Minimal Residual Disease in Patients with Multiple Myeloma by Targeted Tracking Serum M-Protein Using Mass Spectrometry (EasyM). Clinical Cancer Research, 30(6), 1131-1142.
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