By Jennifer Elizabeth Grant, Hong Li
This quantity highlights proteomics reports of quantitative PTM adjustments in either peripheral and important worried method proteomes using the newest advances in mass spectrometry. Chapters comprise sensible details touching on the basics of pattern training, liquid chromatography, and tandem mass spectrometry instrumental research and should elucidate most sensible practices within the interpretation of information utilizing sleek bioinformatics techniques. Written for the preferred Neuromethods series, chapters comprise the type of aspect and key implementation suggestion that guarantees winning leads to the laboratory.
Authoritative and practical, Analysis of Post-Translational variations and Proteolysis in Neuroscience aims to be sure winning leads to the additional research of this important field.
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This quantity highlights proteomics reports of quantitative PTM adjustments in either peripheral and imperative apprehensive process proteomes using the newest advances in mass spectrometry. Chapters contain functional info concerning the basics of pattern coaching, liquid chromatography, and tandem mass spectrometry instrumental research and should elucidate most sensible practices within the interpretation of information utilizing glossy bioinformatics ways.
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Extra resources for Analysis of Post-Translational Modifications and Proteolysis in Neuroscience
5 % Acetic acid in water 4. 1 % TFA in water 5. 5 % Acetic acid in water 6. 5 % Acetic acid in water 7. À80 C freezer (Thermo) 8. Speed-vacuum (Labconco) 34 Haopeng Xiao et al. 5 Boronic Acid Enrichment 1. Boronic acid (BA) conjugated magnetic beads stored in ethanol (BA concentration ¼ 6 mM) 2. 0) 200 mM ammonium acetate buffer 3. Elution buffer: ACN:H2O:TFA ¼ 50:49:1 4. Incubating shaker 5. Magnetic rack 6. À80 C freezer 7. 6 PNGase F Treatment 1. PNGase F (Sigma) 2. ) 3. 7 Glycopeptide Fractionation 1.
These must be removed with a gel-loader tip placed on a P-200 micropipettor; otherwise the solution will not flow through the column efficiently. Always check for appropriate flow. 3. 1 % TFA). 4. 2). 3. NOTE: In rare cases, if the flow rates decrease dramatically upon (or after) loading of sample, the purification procedure can be accelerated by gently applying pressure to the column using the 10 cc plunger after cleaning it with organic solvent. Again make sure to remove air bubbles from the narrow inlet of the column before doing so.
HPLC (Agilent) 3. 6 Â 250 mm 5 μm particle reversed phase column (Waters) 4. 0 in water 5. 8 Stage Tip 1. Stage tips, C18 material 2. 5 % acetic acid 3. Equilibrium buffer: 1 % formic acid (FA) 4. 5 % acetic acid 5. 9 LC-MS/MS Analysis 1. WPS-3000TPLRS autosampler (UltiMate 3000 thermostatted Rapid Separation Pulled Loop Wellplate Sampler, Dionex) 2. Microcapillary column packed with C18 beads (Magic C18AQ, 5 μm, 200 A˚, 100 μm Â 16 cm) 3. 125 % FA 4. 125 % FA 5. Hybrid dual-cell quadrupole linear ion trap-orbitrap mass spectrometer (LTQ Orbitrap Elite, Thermo) 6.