N-terminal Edman Sequencing Sample Preparation

Transcription

1 -terminal dman Sequencing Sample reparation B 2002, ustin, Texas, March 9-12 John eveu, arvard University, Cambridge, M Bill enzel, Genentech nc., South San rancisco, C B 2002, March 8-12, ustin, Texas

2 Objective: Get sample onto the instrument in sufficient purity, quantity and cleanliness to allow successful sequence analysis. Sample is the intact protein(s) or peptide(s) for analysis. nstrument defines the types of supports used for analysis. urity describes the number of proteins or peptides in a sample; assess by various methods including SS-G gel, C. Quantity can be determined by, Bradford/owry/BC assay, gel stain intensity, semi-quantitative C. Cleanliness is a lack of common interfering compounds that could impede the analysis. These five factors will define what types of sample preparation we must do for a successful analysis.

3 Supports for analysis Glass fiber filter, w/polybrene (quaternary polyamine) (B) membrane by electroblotting, filtration, adsorption, and covalent linkage. Bimodal reaction columns (/SX) ()

4 Biobrene / olybrene

5 Common nterferences TS and other amines cause large chemical artifacts. Buffering salts can interfere with the chemistry of the analysis. etergents can affect sample washout from reaction cartridge and conversion flask dynamics. SS can also precipitate in the instrument. ree amino acids contribute to high background in early cycles.

6 Quantitation basics imited by the absolute instrument sensitivity, about 1pmol loaded at ~50% for an average lab. mportant to establish a reasonable level of quantitation for the researcher and the lab. ule: 1ug of 1ka protein = 1 nmol 100ug of 100ka protein = 1,000 pmol 10ug of 100ka protein = 100 pmol 1.0ug of 100ka protein = 10 pmol 0.5ug of 250ka protein = 2 pmol

7 Stain ntensity by mido Black

8 Common Sample reparation Techniques for Soluble Samples irect dsorption to rosorb cartridge eversed-phase (other media) packed micro pipettor tip devices Ultrafiltration membrane cartridge recipitation methods 1 / 2 gel electrophoresis to by electroblotting C separation and collection Micro-spin type cartridges Bimodal reaction cartridges On-bead synthetic peptides Covalent bonding to membrane

9 irect absorption to (and Zitex) membrane Use for samples with high purity. Suitable for samples of reasonably high concentration. llows removal of salts, buffers, free amino acids and some detergents. Sample remains bound to membrane for analysis (addition of polybrene is recommended).

10 rosorb membrane Use for samples with high purity. Can be used with dilute samples. llows extensive washing to remove most contaminants. Sample remains bound to membrane for analysis (addition of polybrene recommended).

11 everse phase (or other media) packed micropipettor-tip devices Suitable for samples of medium to high purity. Can be used with dilute samples, binding to media can be a concern. xtensive desalting, detergent removal possible. nrichment of specific species (i.e.. O4), possible. Sample is eluted in high concentration in solution for analysis or purification.

12 Ultrafiltration membrane cartridge Can be used to remove unwanted low MW components, peptides and proteins from complex mixtures by MW cutoff selection. Can be used with dilute samples. llows extensive washing, buffer exchange, removal of detergents. Sample is eluted in high concentration in solution for analysis or other chemistries.

13 recipitation methods cetone, chloroform/methanol, TC ow to high purity samples. Used to concentrate dilute samples from solution, as little as 50ng total protein. Complete buffer exchange possible. Sample resolubilized in high concentration solution, ideal for loading onto SS-G gel.

14 1 or 2 Gel lectrophoresis lectroblotted to Used for low (1) to very complex mixtures (2) of proteins or peptides. igh sample concentration needed due to limiting gel load volumes. Sample solubility issues may arise. Sample is strongly bound to membrane in a highly concentration for analysis.

15 C separation and collection Suitable for complex mixtures of proteins and peptides. igh sample concentration needed, injection volumes can be limited by column and flow. otential separation and concentration of individual protein and peptide species. Sample is collected in high concentration solution.

16 Capillary C Separation of eptides

17 Micro-spin cartridges for protein and peptide preparation Many new vendors, many chemistries and formats. Uses range from crude preparation of mixtures to final clean up of single analyte for analysis. Match sample with media for optimal results. Multistage processes in convenient formats.

18 ewlett ackard Bimodal reaction columns ery convenient reverse phase sample loading, concentration and washing. On-column chemistries possible, easy clean up.

19 On-bead synthetic peptides Used to confirm synthesis efficiency and correctness. Single beads can contain nanomoles of peptide. n practice, cleavage from the resin is best. andling of single beads is a difficult task to do reproducibly.

20 Covalent binding to Sequelon membrane Chemically bind a single protein or peptide to hydrophobic membrane for analysis. llows efficient extraction of 32 labeled residues, extended sequencing of immobilized proteins, and other special chemistries to be applied. etergents can inhibit efficient binding.

21 Sequelon TC & ryl mine Chemistries

22 Solid hase Sequencing of TC Coupled Betalactogolublin pmol value Cycle

23 eptide Standard: 10pmol (Tyr, la, Glu) 0226B SS 10pmol Standard B SS 10pmol esidue 1 TU S Q T G M TU W T G S Q M W 0226B SS 10pmol esidue 2 TU 0226B SS 10pmol esidue 3 TU G G S Q T M S T Q

24 irty eptide un: o Sequence ata pmol SS irty Boy Standard pmol SS irty Boy esidue 1 S TG TU S Q T G M TU W Q M - - W pmol SS irty Boy esidue 2 S G T TU pmol SS irty Boy esidue 3 S G TU Q M Q T M - W -

25 irect dsorption to Membrane 0301C SS via irect Standard C SS via irect esidue 1 G TU S Q T S G M TU W T M C SS via irect esidue 2 G TU SS via Cycle 2 esidue 3 G TU - S Q T M - S Q T M

26 rosorb ilter Cartridge 0301B SS rosorb Standard B SS rosorb esidue 1 TU Q T S G M TU W T G S Q B SS rosorb esidue 2 TU 0301B SS rosorb esidue 3 TU - S Q G T M - S Q T G

27 C18 Zip Tip Clean Up 0228C SS by Z tip Standard C SS by Z tip esidue 1 TU - Q T S G M TU W - G S T Q M SS by Z tip Cycle 1 esidue 2 TU SS by Z tip Cycle 1 esidue 3 TU G G - S Q T M - S Q T M

28 O, so what if we get no data? nsufficient quantities. ighly heterogeneous sample leading to uninterpretable results. blocked n-terminus, leading into Bill enzel s part of this presentation.