Analytical Gangster Starring Victor Van Deemter

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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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