Increase Phosphopeptide enrichment

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Virginie
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Increase Phosphopeptide enrichment

Postby Virginie » Wed Sep 12, 2012 7:14 am

Hello
I've been doing SILAC phosphoproteomic experiments with poor results. I start with 5mg whole cell lysate that I then fractionate with SCX but end up with 1800 phosphosites. This is the same amount that I get from a single phosphoenrichment of my whole cell lysate (so I know it is not my TiO2 protocol that is at fault). My SCX gradient looks good so I'm wondering if I could loose my peptides by overdrying them in the speed vac after the SCX, or after the Oasis C18 desalting step?
Does anyone have any ideas?
Thanks.

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Doug
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Postby Doug » Wed Sep 12, 2012 8:07 am

Is it possible that you are overloading the TiO2 beads/column? If so doing the enrichment after SCX should help. I have always done phosphopeptide enrichment after SCX and it works quite well. It's a little more work but might be worth it.

Just out of curiosity, what percentage of your IDs or phosphorylated vs non-phosphorylated?

Virginie
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Postby Virginie » Wed Sep 12, 2012 1:34 pm

I do the phosphoenrichment after the SCX. I get from about 70 to 90% phosphorylated peptides depending which fraction of the SCX I'm looking at (the low KCl have a higher percentage than the higher concentration of KCl)

Infinity
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Postby Infinity » Thu Sep 13, 2012 3:22 pm

Well, since you have high enrichment level and low number of identified phosphopeptides at the same time, - than it might be something wrong with your LC-MS/MS runs (sensitivity, quality of LC, calibration). I would look at the 1. QC runs from the same day (if there are any); 2. Chromatogramms (Intensity, peak width); 3. mass errors for identified peptides. Unfortunately only in case of the bad calibration you will be able to recalibrate you raw files offline instead of repeating the whole experiment.
If everything was fine with MS, then it would be really helpful posting here your entire detailed protocol.

Virginie
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Postby Virginie » Fri Sep 14, 2012 5:19 am

The QC runs of the same day (BSA 80% coverage) look good. The chromatograms have a TIC of 10x8 with a small peak width and the mass error is in general under 10ppm.
I'll write my protocol in a bit but it was adapted from a paper from M. Mann.

Virginie
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Postby Virginie » Fri Sep 14, 2012 7:04 am

Cell lysis in urea
Digest 5mg with trypsin
Desalt with SepPak
Lyophilize
Resuspend powder in buffer A for SCX (5mM KH2PO4 pH 2.7, 25% ACN)
Collect 20 fractions on SCX (column polysulfyl A)
Buffer B: 5mM KH2PO4 pH 2.7, 500mM KCl, 25%ACN
Speed Vac to dryness the fractions
Desalt with Oasis column
SpeedVac to dryness
Enrich the fractions with TiO2 columns (PIERCE)
Desalt the fractions with graphite columns (PIERCE)
SpeedVac to dryness
Resuspend in 0.1% FA
Run on Orbi same day

AAlpert
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Postby AAlpert » Fri Sep 14, 2012 7:20 am

Here's a couple of things to consider:

1) Desalting the TiO2 fractions with graphite: Graphite acts like an affinity material regarding phosphopeptides. You can elute singly phosphorylated peptides with conditions similar to those with C-18 (60-70% ACN containing 0.1% formic acid or some such) but those conditions do not suffice to elute peptides with more than one phosphate. For those you need to replace the formic acid with 200 mM NH4OH or (better) 200 mM pyrrolidine. See my poster from ASMS 2007 (in the Literature section of the PolyLC web site).

2) Per Doug's suggestion: If you have a few phosphopeptides of much higher abundance than the rest, then they could be displacing the low-abundance phosphopeptides if the capacity of the TiO2 cartridge is marginal for retaining them all (per my poster from ABRF 2004; same Literature section) and you'll get a nonrepresentative sampling. Try collecting the filtrate from the TiO2 cartridge and passing it through a second TiO2 cartridge. If you identify phosphopeptides from the second cartridge, then you'll know that you've exceeded the capacity of the first cartridge. This would be a problem in particular with the first few fractions from the PolySULFOETHYL A column, in which one finds about 30% of the phosphopeptides from a typical complex tryptic digest.

Virginie
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Postby Virginie » Mon Sep 17, 2012 7:32 am

Thanks
I'll try both suggestions.

tentionfree
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Postby tentionfree » Thu Jan 24, 2013 4:17 am

Dear you identify phosphopeptides from the second cartridge, then you'll know that you've exceeded the capacity of the first cartridge. This would be a problem in particular with the first few fractions from the PolySULFOETHYL A column, in which one finds about 30% of the phosphopeptides from a typical complex tryptic digest.
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