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
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
