100% satisfaction guarantee Immediately available after payment Both online and in PDF No strings attached
logo-home
Previously searched by you
Previously searched by you
logo
Summary Concepts of Protein Technology and Applications (14/20) R218,81
Add to cart
Quickly navigate to
Preview
  2.  
  3. Universiteit Antwerpen (UA)
  4.  
  5. Biomedische Wetenschappen
  6.  
  7. Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)

Summary

Summary Concepts of Protein Technology and Applications (14/20)
 174 views  11 purchases
  • Course
  • Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)
  • Institution
  • Universiteit Antwerpen (UA)

This document serves as a comprehensive summary combining the key insights from the presentations of Professors Xaveer van Ostade and Kurt Boonen. The summary consists of their slides, enriched with my personal notes.

Last document update: 11 months ago

Preview 4 out of 155  pages

Document information

  • December 18, 2023
  • December 28, 2023
  • 155
  • 2023/2024
  • Summary

Subjects

  • concepts
  • proteomics
  • mass spectrometry
  • 1st master
  • xaveer van ostade
  • kurt boonen
  • ms

Written for

  • Universiteit Antwerpen (UA)
  • Biomedische Wetenschappen
  • Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)
All documents for this subject (23)

Seller

avatar-seller
BMWstudent19

Reviews received

Content preview

CONCEPTS OF PROTEIN TECHNOLOGY
AND APPLICATIONS
INHOUDSOPGAVE

1.1 Definition proteomics .......................................................................................................................... 1

1.2 Why proteomics? ................................................................................................................................ 1
1.2.1 Compared to genomics, proteomics is “the real thing” ....................................................................... 1
1.2.2 mRNA vs protein profiling ................................................................................................................... 2
1.2.3 More (6-8) proteins/gene .................................................................................................................... 2
1.2.3.1 Posttranslational modification ........................................................................................................ 2
1.2.3.2 Alternative splicing à isoforms ...................................................................................................... 3
1.2.4 Protein interaction networks ............................................................................................................... 3
1.2.5 Cellular localization ............................................................................................................................. 3

1.3 Proteomics as part of systemsbiology .................................................................................................. 4

1.4 The different faces of proteomics ........................................................................................................ 4

1.5 Identification of proteins: principles .................................................................................................... 5
1.5.1 Sample preparation ............................................................................................................................. 6

1.5.2 Protein peptide separation .................................................................................................................. 6
1.5.2.1 Increasing separation capacity (peak capacity) ............................................................................... 7
1.5.2.2 2D-Electrophoresis (2D-PAGE) ........................................................................................................ 7
1.5.2.3 Chromatography ............................................................................................................................. 8
1.5.3 Analysis: mass spectrometry ............................................................................................................... 9
1.5.4 Protein identification by data analysis .............................................................................................. 10

1.6 Workflows for ‘shotgun’ proteomics .................................................................................................. 10

2.1 Proteomics introduction .................................................................................................................... 11

2.1.1 Part of life sciences ............................................................................................................................ 11
2.1.2 from genome to proteome ................................................................................................................ 12
2.1.2.1 Why proteomics? .......................................................................................................................... 12
2.1.2.2 The proteome is very complex! ..................................................................................................... 12
2.1.2.3 From genome to proteome ........................................................................................................... 13
2.1.3 Protein variants in disease................................................................................................................. 13

2.2 Overview: what do we do with a complex sample? ........................................................................... 14


1

,2.3 Sample preparation steps .................................................................................................................. 15

2.3.1 Defining your research question ........................................................................................................ 16
2.3.2 Literature study ................................................................................................................................. 16

2.3.3 Sample collection............................................................................................................................... 17
2.3.3.1 Sample types ................................................................................................................................. 17
2.3.3.2 Sample degradation ...................................................................................................................... 17
2.3.4 Protein extraction – solubilization ..................................................................................................... 17
2.3.4.1 Protein extraction.......................................................................................................................... 17
2.3.4.2 Protein solubilization ..................................................................................................................... 20

2.3.5 Protein separation or purification ..................................................................................................... 22
2.3.5.1 Acetone precipitation .................................................................................................................... 22
2.3.5.2 Gelfiltration ................................................................................................................................... 22
2.3.5.3 Filtering.......................................................................................................................................... 22
2.3.5.4 SDS-PAGE....................................................................................................................................... 23
2.3.6 Reduction and alkylation ................................................................................................................... 23
2.3.7 Digestion............................................................................................................................................ 24
2.3.8 Peptides purification or selection ...................................................................................................... 25
2.3.9 Prefractionation ................................................................................................................................ 26

3.1 Introduction ...................................................................................................................................... 27

3.1.1 AA analyses and AA sequencing are two different techniques .......................................................... 27
3.1.2 What can Amino Acid Analysis (AAA) do for you? ............................................................................. 27

3.2 Sample Preparation for Amino Acid Analysis ..................................................................................... 28

3.3 AAA of proteins/peptides .................................................................................................................. 28

3.3.1 In general........................................................................................................................................... 28
3.3.2 Hydrolysis .......................................................................................................................................... 28

3.3.3 Separation of amino acids ................................................................................................................. 30
3.3.3.1 Paradigms ...................................................................................................................................... 30
3.3.3.2 Pre-column derivatization ............................................................................................................. 30
3.3.3.3 Post-column derivatization ........................................................................................................... 31
3.3.3.4 Pre- versus post-column derivatization ......................................................................................... 32

3.4 Amino acid sequencing ...................................................................................................................... 33
3.4.1 In general........................................................................................................................................... 33
3.4.2 Edman degradation ........................................................................................................................... 33
3.4.2.1 Insulin ............................................................................................................................................ 33
3.4.2.2 How does it work? ......................................................................................................................... 33
3.4.2.3 Cyclical estimation of yields .......................................................................................................... 34
3.4.2.4 Side products ................................................................................................................................. 34
3.4.2.5 Edman chemistry also disrupts AAs............................................................................................... 35
3.4.2.6 Positive aspects and drawbacks of Edman sequencing ................................................................. 35



2

, 3.4.3 Cleavage ............................................................................................................................................ 35
3.4.3.1 Chemical cleavage ......................................................................................................................... 35
3.4.3.2 Enzymatic cleavage ....................................................................................................................... 36

3.5 Carboxyterminal sequencing ............................................................................................................. 36
3.5.1 Carboxypeptidases ............................................................................................................................ 36
3.5.2 Chemical process ............................................................................................................................... 36

3.6 Single molecule sequencing ............................................................................................................... 37
3.6.1 Edman degradation ........................................................................................................................... 37
3.6.2 Microscale Edman degradation......................................................................................................... 37

4.1 Introduction ...................................................................................................................................... 39
4.1.1 Electrophoresis .................................................................................................................................. 39
4.1.2 2-dimensional gel electrophoresis ..................................................................................................... 39

4.2 Basic principles .................................................................................................................................. 39

4.2.1 In general........................................................................................................................................... 39
4.2.2 In practice .......................................................................................................................................... 40

4.3 First dimension: Isoelectric focusing (IEF)........................................................................................... 40
4.3.1 Isoelectric focusing ............................................................................................................................ 40
4.3.2 Protein charge ................................................................................................................................... 40
4.3.3 Creating a pH gradient for IEF ........................................................................................................... 41
4.3.3.1 Using carrier ampholytes .............................................................................................................. 42
4.3.3.2 IMMOBILIZED PH GRADIENTS ....................................................................................................... 42

4.3.4 2D-GE sample preparation ................................................................................................................ 43
4.3.5 Standard IP run for IEF and 2D-GE..................................................................................................... 44

4.4 Second Dimension: SDS-PAGE............................................................................................................ 44
4.4.1 SDS-PAGE ........................................................................................................................................... 44

4.4.2 Mass markers .................................................................................................................................... 45
4.4.3 PAGE matrix....................................................................................................................................... 46
4.4.3.1 Composition .................................................................................................................................. 46
4.4.3.2 Polymerization............................................................................................................................... 46
4.4.4 SDS-PAGE systems ............................................................................................................................. 46

4.5 Sample preparation ........................................................................................................................... 47
4.5.1 IEF preparation .................................................................................................................................. 47
4.5.2 Preparation of pH gradient gels ........................................................................................................ 47
4.5.3 IPG preparation for second dimension .............................................................................................. 47




3

, 4.6 Protein detection methods 2D-GE...................................................................................................... 48

4.6.1 Coomassie Brilliant Blue (CBB R-250) ................................................................................................ 48
4.6.2 Silver staining .................................................................................................................................... 49

4.6.3 SYPRO Ruby staining.......................................................................................................................... 49
4.6.4 2D-GE autoradiography..................................................................................................................... 49
4.6.5 Overview of staining options ............................................................................................................. 49

4.7 Analysis of 2D-gels ............................................................................................................................. 50
4.7.1 Image processing – digitalization ...................................................................................................... 50
4.7.2 Protein spot or ‘gradu’....................................................................................................................... 50

4.7.3 Complexities of 2D-GE ....................................................................................................................... 50

4.8 Differential in-gel electrophoresis (DIGE) ........................................................................................... 51
4.8.1 In general........................................................................................................................................... 51
4.8.2 Application ........................................................................................................................................ 51

4.8.3 Example ............................................................................................................................................. 52
4.8.4 DeCyder v. 6.5.................................................................................................................................... 52

4.9 2D-GE protein identification .............................................................................................................. 52

4.10 Take home points .............................................................................................................................. 52

5.1 Introduction ...................................................................................................................................... 53

5.2 Hydrophobic interactions .................................................................................................................. 53

5.3 Standard conditions for RP-HPLC ....................................................................................................... 54
5.3.1 Stationary phase................................................................................................................................ 54
5.3.2 Mobile phase ..................................................................................................................................... 55
5.3.2.1 Organic solvent .............................................................................................................................. 56
5.3.2.2 pH of mobile phase ....................................................................................................................... 58
5.3.2.3 Agents for ion-pairing .................................................................................................................... 58

5.4 Microcolumn Reverse Phase chromatography ................................................................................... 60
5.4.1 General .............................................................................................................................................. 60
5.4.2 Principle ............................................................................................................................................. 60

5.4.3 Adaptation of optimal flow rate ........................................................................................................ 61
5.4.4 Capacity and resolution of microcolumns ......................................................................................... 61
5.4.5 Adaptations of the HPLC system ....................................................................................................... 62

5.5 2D-LC ................................................................................................................................................. 63
5.5.1 Peak capacity and set-up of a 2D-LC ................................................................................................. 63



4

The benefits of buying summaries with Stuvia:

Guaranteed quality through customer reviews

Guaranteed quality through customer reviews

Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.

Quick and easy check-out

Quick and easy check-out

You can quickly pay through EFT, credit card or Stuvia-credit for the summaries. There is no membership needed.

Focus on what matters

Focus on what matters

Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!

Frequently asked questions

What do I get when I buy this document?

You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.

Satisfaction guarantee: how does it work?

Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.

Who am I buying this summary from?

Stuvia is a marketplace, so you are not buying this document from us, but from seller BMWstudent19. Stuvia facilitates payment to the seller.

Will I be stuck with a subscription?

No, you only buy this summary for R218,81. You're not tied to anything after your purchase.

Can Stuvia be trusted?

4.6 stars on Google & Trustpilot (+1000 reviews)

56326 documents were sold in the last 30 days

Founded in 2010, the go-to place to buy summaries for 14 years now

Start selling
R218,81  11x  sold
  • (0)
Add to cart
Added