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Sample preparation. Sample Preparation Voyager Training Class.

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Presentation on theme: "Sample preparation. Sample Preparation Voyager Training Class."— Presentation transcript:

1 Sample preparation

2 Sample Preparation Voyager Training Class

3 Sinapinic AcidProteins >10kDa a-Cyano-4-hydroxy-cinnamic acid (CHCA) Peptides<10kDa 2,5-Dihydroxybenzoic acid (DHB) Neutral Carbohydrates, Synthetic Polymers “Super DHB”Proteins, Glycosylated proteins Oligonucleotides HABAProteins, Oligosaccharides 3-Hydroxypicolinic acid Matrix selection

4 Vacuum dried Milky, amorphous Air dried Irregular crystals in ring DHB Crystallization

5 DNA Analysis

6  - cyano Sinapinic acid Rounded Rhomboid-shaped Appearance of Matrix

7 Sample Plates 1 mm diameter Ability to load 1µl sample and matrix Application: Enhanced sensitivity by concentrating sample; useful in high throughput methods

8 Advanced Sample Preparation Voyager Training Class

9 Sample Dilution/Concentration Note: highly dilute samples can be concentrated by Speed- Vac or Solid Phase Extraction. Compound Concentration 0.1 to 10 pmol/µL 10 to 100 pmol/µL Peptides and proteins Oligonucleotides Polymers 100 pmol/µL Dilute samples to the concentrations shown in the table below. If the sample concentration is unknown a dilution series may be needed to produce a good spot on the MALDI plate.

10 Drop dialysis cleanup of Enolase Yeast Enolase (47 kDa) in 8 M urea was dialyzed for 1 hr on a Millipore membrane. After Before

11 ConditionCondition the ZipTip with 10 µl of acetonitrile (ACN), then 10 µl of 50% ACN/0.1% TFA, then 2 x 10 µl of 0.1% TFA. LoadLoad the sample onto the ZipTip by pipetting 5-10 µl sample up and down several times and discarding the liquid. WashWash C 18 tip with 3 x 10 µl of 0.1% TFA to remove salts. EluteElute the sample from the ZipTip with 30-70% ACN or elute directly into the matrix (e.g. CHCA in 50% ACN/0.1%TFA); minimal volume of ~3 µl can be used. Procedure for using C 18 ZipTips

12 Use of the C 18 ZipTip 1.Sample Concentration and Buffer Removal Dilute samples can be concentrated by adsorbing analyte from multiple 10  l aliquots into the ZipTip and eluting out into a small volume, effecting a 10- to 50-fold concentration. Mild conditions (e.g. 0.1% TFA) will retain peptides and proteins on a ZipTip but remove common buffers and salts such as: 2M NaCl, 100mM Phosphate, 8M Urea, 6M Guanidine or 50% Glycerol For more detailed information on ZipTips, click hereclick here

13 C 18 Efficiently Removes digestion Buffer -40000 -20000 0 20000 40000 Counts 1000 1500 2000 2500 3000 3500 4000 4500 Mass (m/z) C18 Prep in PBS/Urea/NaCl Standard Prep in PBS/Urea/NaCl Analysis of a peptide map of IgG HC digest containing phosphate, NaCl, urea and DTT at 0.1 mg/ml digested with endo Lys C.

14 Protein Sequencing of Myoglobin using In-Source Decay -Leu/Ile(113.271) -Phe(147.295) -Thr(101.414) -Gly(56.234) -?(234.912) -Glu(128.776) -Thr(101.183) -Leu/Ile(113.112) -Glu(129.24) -Gln/Lys(127.786) -Phe(147.783) -Asp(114.958) -Gln/Lys(128.387) -he(146. P496) -Gln/Lys(128.174) -His(136.746) -Leu/Ile(113.488) 5575.102 3500 4000 4500 5000 5500 Ref:V.Katta,70,4410-4416

15 Proteomics Voyager Training Class

16 In-Gel Digest Fundamentals Handling the gel and slices Washing and destaining Enzymatic Digestion Peptide Extraction Concentration/Cleanup MALDI-TOF Analysis

17 In-Gel Digest Method MALDI-TOF Analysis Acquire a good spectrum in reflector mode with a method optimized for high resolution in 800-3000 Da range. Calibrate with internal Trypsin peaks T7 (842.5099) and T4 (2211.1046) if present, otherwise use close external calibration. Alternatively, samples can be spiked with dilute Cal Mix 1 or 2 (approx. 1:500 in the matrix) for internal calibration. Finally, samples can be internally re-calibrated with known peak masses from a good Protein Prospector MS-Fit hit. If spectrum is poor due to contaminants or low peptide concentration try cleanup and/or concentration of the remaining sample with ZipTip C 18

18 Nanotechnology - carbon clusters

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