Thanks Dick!
on a side note, reading those papers from Brian,I am pretty skeptical that
the amount people are loading is actually what they are loading. It was
really an eye opener when I started to check my digest concentrations using
AAA. Digestion efficiency can be all over the place and BCA, bradford etc
to not work well with peptides. Thermo's new fluorescent peptide assay
actually seems pretty decent...which i'm happy about
Rest of post
On Fri, Sep 25, 2015 at 12:10 PM Richard F Cook <email obscured>> wrote:
> Brett,
>
> A quick rule for deciding how much to load on different diameter reverse
> phase columns is “ (D1/D2) squared “. [D1 being the bigger diameter and D2
> the smaller].
>
> So if you are OK with loading 1 mg on a a 4.6 mm diameter column and want
> to know how much you can load on a 75 um…the above gives you 3761…so you
> would want to load 1/376 th of 1 mg onto the 75 um column…all other things
> being equal ... That should get you in the ball park...
>
> Best,
>
> Dick
>
>
> > On Sep 25, 2015, at 2:13 PM, Brett Phinney <email obscured>> wrote:
> >
> > Thanks Brian! I didn't know about that 2015 paper!!
> >
> > On Fri, Sep 25, 2015 at 10:52 AM Brian Hampton <email obscured>>
> wrote:
> >
> >> Hi Brett,
> >>
> >> I recall more than one paper that addresses this, the one you are
> looking
> >> for is probably not any of the below since they are fairly recent, but
> do
> >> address your question.
> >>
> >> "Systematical optimization of reverse-phase chromatography for shotgun
> >> proteomics"
> >> http://dx.doi.org/10.1021/pr900251d
> >>
> >> "Off-line two-dimensional liquid chromatography with maximized sample
> >> loading to reversed-phase liquid chromatography-electrospray ionization
> >> tandem mass spectrometry for shotgun proteome analysis."
> >> http://dx.doi.org/10.1021/ac802106z
> >>
> >> "Systematic optimization of long gradient chromatography mass
> spectrometry
> >> for deep analysis of brain proteome."
> >> http://dx.doi.org/10.1021/pr500882h
> >>
> >> Brian Hampton
> >> Protein Analysis Lab
> >> Center for Vascular and Inflammatory Diseases
> >> University of Maryland School of Medicine
> >> 655 West Baltimore Street BRB 7-018
> >> Baltimore MD 21201
> >> V: 410-706-8207
> >>
> >>
> >> On Fri, Sep 25, 2015 at 1:10 PM, Brett Phinney <email obscured>>
> wrote:
> >>
> >>> Ok so a few months ago I came across a paper or technical note, or
> maybe
> >> I
> >>> just dreamt it who knows...and now for the life of me I can't find it
> >>>
> >>> anyway it was about the optimum loading capacity of a C18 75um column
> and
> >>> how over loading a column will lead to decreased results (id's and
> quant)
> >>> after a set point. This is all obvious of course, but I was wanting to
> >> see
> >>> what they determined in their hands.
> >>>
> >>> In our case loading anything above 1 ug or so seems to be the breaking
> >>> point for us using our columns, although getting an accurate protein
> >>> concentration on digested peptides is challenging and my measurements
> >> could
> >>> be way off a lot of the times. Speaking of which I need to dig up my
> >>> comparison of thermo's new florescent peptide assay with our
> traditional
> >>> AAA....It's not an idea comparison due to vastly different starting
> >> amount
> >>> requirements but at least it's something
> >>>
> >>> Anyway if anyone has seen such a paper I'd be very appreciative!
> >>>
> >>> Thanks!!
> >>>
> >>> Brett
> >>>
> >>> ――
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