Memorial Sloan-Kettering Cancer Center
This workshop started with short presentations by Paul Tempst, David Speicher (Wistar Institute) and Arie Admon (UC Berkeley). A brief survey among the participants indicated that most people are usually dealing with sequencing runs shorter than 30 residues and almost never over 50 cycles. The term "micro-sequencing" was widely interpreted at first (i.e. from 2 to 20 picomoles initial yield) but after some discussion, most agreed that it represents the highest sensitivities obtained with an off-the-shelf instrument. Those sensitivities are well below 10 picomoles. Clearly, the major interests of the participants concerned this sensitivity issue as well as sample preparation and handling prior to sequencing.
It was reported that losses of proteins and peptides on glass and plastic (e.g. eppendorf tubes etc.) are on the order of 40 - 50%, following 15 sec to 1 min of storage under dilute conditions (10 picomoles in 30-100 ul). Remedies for sample losses consist of getting proteins immobilized as early as possible (e.g. cell extracts to 2D gels to membrane) or to supplement peptide solutions with acid before transfer. It was found that adding neat TFA in a 1 to 4 ratio (TFA/sample; vol/vol) after storage, just before transfer (e.g. to the sequencer cartridge), increases peptide recoveries from 55% to 85%. Several participants had interesting stories about sources of polypeptide contamination (e.g. synthetic peptides, paper from dot matrix printers, powder from gloves, etc.) that create enormous problems when sequencing at low picomole levels. The obvious conclusion was that final stages of sample prep and sequencing should be done in a dedicated area that is kept scrupulously clean.
There was much interest in improving the sensitivity of sequencing PVDF-blotted samples. According to David Speicher, and concurred to by several participants, the best results with sequencing off PVDF are obtained by pulsing solvents during washes and extraction, delivering TFA as a vapor (with bubbling through the liquid TFA in the reagent bottle), extraction with heptane/ethyl acetate (1:1 v/v) due to the superior PVDF-wetting properties of heptane (1) and use of the cross-flow blot cartridge (2). If this is combined with injection of a large portion of the PTH's in the flask (>70%), sequencing at subpicomole levels becomes feasible (3,4). Accurate injections of as much as 80% of the sample are facilitated by putting an additional restrictor in the injector assembly (in the loop).
A consensus emerged that for femtomole level PTH analysis, baseline drift and chemical background should be minimized. The former can be accomplished by titrating the baseline by addition of tryptophan or acetone to solvent A; in this way, analysis at 0.001 AUFS (269 nm) is possible. Titration is done after a standard chromatogram has been run; based on the slope of the baseline, adjustments are then made as necessary (3). Chemical background can be reduced by using a smaller cartridge (15 ul volume); however, no exact figures were available. Other ways are more frequent changes of reagents (weekly) and cleaning of the instrument (every other week) and by reducing PITC concentrations (down to 0.5 % ) and TMA pressure (down to 0.3psi) which, in combination, reduce background to one third (approximately 1 picomole of DPU). Reducing the amount of polybrene cuts background but also results in increased washout of peptides at the 5 to 10 picomole level (78 % washout of a 25-mer after 15 cycles using 1.5 mg polybrene, as compared to 56% washout from 3 mg polybrene) (4). Finally, it was suggested that for micro peptide sequencing, 5-10 picomoles of a specifically designed synthetic peptide be used as a standard, to evaluate initial and repetitive yields, instead of mega-amounts of B-lactoglobulin.
References:
1. Speicher, D.W. (1989) in Techniques in Protein Chemistry (Hugli, T.E. ed) pp 24-35, Academic Press, New York.
2. Reim, D.F., Hembach, P. and Speicher, D.W. (1992) in Techniques in Protein Chemistry 111 (Angeletti, R. ed) pp 5360, Academic Press, New York.
3. Tempst, P. and Riviere, L. (1989) Anal. Biochem. 183:290-300.
4. Erdjument-Bromage, H., Geromanos, S. and Tempst, P. (in press) in Techniques in Protein Chemistry IV (Hogue Angeletti, R. ed) Academic Press, New York.
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