1. Sample preparation Protein and peptide separation techniques Karel Bezstarosti (Proteomics Center, Erasmus MC)

2. Proteomics Identify all proteins present in a sample.
Characterize the post-translational modifications (PTM’s)of these proteins.
Protein quantification.

3. Sample preparation for proteomic analysis
No universal protocol for sample preparation.
Method used depends on:
Sample type: Cultured cells
Tissue
Body fluid
Analytical techniques
used and goal of Global proteomic analysis
experiment: Expression analysis
Targeted analysis.
Amount of sample. PTM analysis

4. Sample preparation for proteomic analysis
Workflow:
Sample collection
Stabilization
Protein extraction
Improving Subcellular fractionation
detectability Abundant protein depletion
Enrichment of protein groups
Protein fractionation
Analyze proteins Reduction
Alkylation
tryptic digestion
contaminants removal
Mass spec analysis

5. Sample preparation for proteomic analysis
Sample collection and stabilization:
Protect proteins from degradation and modification
Cultured cells: During sample collection keep sample as cold as possible.
Use protease and phosphatase inhibitors in solutions
Inactivate enzym activity by heat (>80OC)
Tissue: Snap freeze sample in liquid nitrogen.
Protect protein sample from contamination(Keratins, Detergent ……)
Wear gloves when handling samples

6. Sample preparation for proteomic analysis
Protein extraction cell lysis:
Gentle lysis (release of organelles)
osmotic shock (Cultured cells..)
handheld homogenizer or blender(liver, brain..)
(detergent)
Moderate lysis:
blade homogenizer(most animal tissues)
grinding frozen tissue sample with mortar and pestle
Vigorous lysis:(full homogenization)
ultrasonication
french press

7. Sample preparation for proteomic analysis
Protein solubilization
The chosen solubilization strategy should be compatible with other steps in the workflow
Solubilize as many proteins as possible
All interactions with other proteins, lipids, or nucleic acids need to be disrupted.
Prevent degradation or modification of proteins.
high reproducibility

8. Sample preparation for proteomic analysis
Commenly used buffers:
Ammonium bicarbonate (in-gel tryptic digestion)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
2-Amino-2-(hydroxymethyl)propane-1,3-diol(TRIS)
25-50 mM is usually sufficient to maintain pH.
Buffer additives:
Protease and phosphatase inhibitors
(Salts)
Detergents

9. Sample preparation for proteomic analysis
Detergents
SDS
SDC
Triton-X100
Chaps
Etc.
Enhance solubility of target proteins(membrane proteins).
Can improve tryptic digestion.
Are usually bad for LC-MS.
There are some detergents that are LC-MS compatible.
Rapigest SF(Waters) and PPS Silent surfactant(Expedeon)
Acid-labile surfactants are hydrolyzed at low pH, and the hydrolysis products are compatible with reversed-phase separations and MS.

10. Sample preparation for proteomic analysis
Workflow:
Sample collection
Stabilization
Protein extraction
Improving Abundant protein depletion detectability Subcellular fractionation Enrichment of protein groups
Protein fractionation
Analyze proteins Reduction
Alkylation
tryptic digestion
contaminants removal
Mass spec analysis

11. Challenges in proteomic sample preparation
Protein sample complexity and dynamic range.
dynamic concentration range amount of proteins
Cultured mammalian cells >104
Tissue(multiple cell types) >104
Plasma >106
Mass spectrometers dynamic range:

12. Plasma Proteome Dynamic range

13. Plasma proteins

14. Depletion of Abundant Plasma Proteins
Agilent MARS-14 depletion Column

15. Subcellular fractionation using differential centrifugation

16. Complex Immunoprecipitation (Co-IP)

17. SDS-PAGE 1D gel electrophoresisMW
Marker

18. 2D Gel Electrophoresis

19. Rat (LV)Heart ProteinsMW
100000
5000 pH

20. Sample preparation for proteomic analysis
Workflow:
Sample collection
Stabilization
Protein extraction
Improving Abundant protein depletion detectability Subcellular fractionation Enrichment of protein groups
Protein fractionation
Analyze proteins Reduction Alkylation
tryptic digestion
contaminants removal
Mass spec analysis

21. Bottom-up proteomics: Proteins are first digested into peptides.
Advantages:
The mass spectrometer is most efficient at obtaining sequence information from peptides.
Fragmentation of tryptic peptides is very well understood.
Sensitivity of the mass spectrometer for peptides is much higher than for proteins.
Separation of peptides using simple mass spec friendly buffers is much easier than for proteins.
Data acquisition and data processing is highly automated.

22. Bottom-up proteomics: Proteins are first digested into peptides.
Disadvantages:
Relationship between peptide and protein is lost.
Already complex protein sample will be up to 100x more complex.

23. Sample preparation for proteomic analysis

24. Trypsin
Very specifically cleaves proteins on the carboxy-terminal side of Lysine(K) and Arginine(R) residues.
CCSDVFNQVVK SISIVGSYVGNR ADTR EALDFFAR GLVK
Produces peptides with a basic C-terminus. These peptides give good (CID) fragmentation spectra.
Other proteases
Lys C
Arg C
Asp N
-----KCCSDVFNQVVKSISIVGSYVGNRADTREALDFFARGLVK-----

25. Proteomics workflow (Bottom-up)
Protein mixture
Peptide mixture
NanoLC
etc

26. Data dependent LC-MS/MS analysis (DDA)
TIC
Full MS scan
MS/MS scan
(30x)
1 cycle ~ 1 sec

27. Amino acid sequence determination
etc Y F A I E

28. Multidimensional Liquid Chromatography (for very complex peptide mixtures)
First dimension of separation is different from the separation in the second dimension that is hydrophobicity(Reversed Phase).
Strong Cation Exchange(charge) - Reversed Phase
Strong Anion Exchange(charge) - Reversed Phase
Reversed Phase High pH Reversed Phase
HILIC Reversed Phase
…… Reversed Phase
1st dimension
2nd dimension MS

29. HILIC (hydrophilic interaction liquid chromatography)
1st dimension
2nd dimension

30. HILIC fractionation and LC-MS
HILIC (hydrophilic interaction liquid chromatography)
HILIC fractionation and LC-MS
# Protein IDs depends on MS speed and sensitivity, sample concentration, sample complexity, LC gradient length, analysis time, extent of fractionation, etc. 10 20 30 40 50 60 70 80 90
100
110
Time (min)
So the result of an analysis in terms of protein identifications depends on a number of factors like fractionation, MS speed and sensitivity, sample concentration, LC gradient length, measurement time, and so on. The standard procedure in the lab when dealing with complex mixtures these days is peptide fractionation using HILIC chromatography followed by 2hr gradients on an LC-MS setup. This way, we can routinely identify >6000 proteins from a HeLa cell lysate of 100 ug total protein in less than 16 hrs.
>8,000 proteins in a HeLa cell lysate identified in <16 hrs

31. Thank you Questions? http://www.proteomicscenter.nl/
Please feel free to contact us so that we can help you design your experiments.
Thank you
Questions?