Analytical Gangster Starring Victor Van Deemter
True Speak
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Size exclusion size exclusion leave my mind confused when Monomer and dimer need better resolution You've got a mab therapeutic for breast cancer And you have to quantitate the aggregate in the sample Of course LC is preferred but in this specific instance It should be run under non-denaturing conditions To do this, is through this, size exclusion separation This way you can assess the extent of aggregation SEC is a nonadsorptive separation mode Selectivity by how molecules enter and exit pores Retention is based on hydrodynamic volume It might seem simple but there are often problems Since its non-adsorptive, it's driven by entropy To separate the monomer from aggregated entities But based upon the mobile phase and bonded chemistry Adsorption and secondary interactions can affect the peaks So we bond a silane like a diol that's hydrophilic Basic residues on the protein don't interact with the acidic Silanols or peaks tail and retention times change Because we don't have optimal bonding of silanes Too hydrophobic a phase, I've now got a HIC column Running with high salt so this can be a big problem So one way to minimize the interaction and to solve it Is to modify the MP with 10% organic solvent A protic modifier like IPA is most common Address the stickiness hydrophobic problem So why is SEC important, well the FDA is asking you To characterize proteins by critical quality attribute Certainly a 5µm column would suffice Back in the day when LC systems weren't this nice Sub-2um SEC columns nowadays is common But SEC is non-adsorptive so extra column volume Is more pronounced, linear velocities are low so And backpressures aren't that high because of the low flow A super tight system is needed for the whole So Extraneous tubing and large flows cells is a no-no When comes to SVA you'll never see me stressed Cause when it comes to peptide maps they call me GPS To characterize protein, one of the only ways Is an enzymatic digest with a serine protease Trypsin it is, probably the most common Then run tryptic peptides on reversed phase columns First denature the protein with heat and chaotrope Though MS-friendly detergents might be the way to go Urea may carbamylate, could be an issue So guanidine HCl may be what you wanna stick to Next is TCEP or DTT but a forewarning You'll need to alkylate to prevent disulfides reforming Now digest with trypsin, maybe overnight To keep digest consistent so you know it's right Although Trypsin cleaves n terminal arg and lysine A lot of this has to do with the digestion timing Too short and you might see a missed cleavage Subsequently less coverage of the primary sequence Digestion too long, non trypsin activity So no longer that the trypsin specificity specifically Chymotrypsin cleavage might be seen this way So use methylated trypsin and TPCK MS grade trypsin definitely the way to go But keep in mind enzyme to substrate ratio You don't want to introduce enzymatic artifacts You'll miss a target mass and you don't wanna argue that Deamidation especially asparagines near glycines Succinimide formation and iso asp is what I've seen Likely because digestion buffer is too basic If that's the case then, you need to make a few changes Like digestion at lower pHs or a low temp Or shorten the digestion time, might make more sense Notice the algorithms bioinformatics uses Have to confirm the b and y ions or it's useless So confirm the MS/MS for the proof it's Not a false positive that your software is producing
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