Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

Подождите немного. Документ загружается.

3 Mass Spectrometry270

Fig. 3.33: The workflow for DeCyder MS. DeCyder MS uses

data derived from an electrospray MS experiment.

Fig. 3.34: DeCyder MS PepMatch module (matching module)

showing the different expression of one particular peptide in

three repeats (courtesy of A. Parbel GE Healthcare).

3.6 MS Strategies 271

Fig. 3.35: Comparison of traditional LC-MS profile to that of

DeCyder MS, whereby peptide components can be clearly detected

with DeCyder MS. This visualization of an LC-MS profile in this

fashion is useful for a range of troubleshooting and monitoring needs.

(courtesy of A.Parbel GE Healthcare).

3.6

MS Strategies

Effectively there are two strategies, bottom up and top down. The dif-

ference, essentially, is what is the state of the sample when it enters

the mass spectrometer i.e. is it a peptide or a protein?

3.6.1

Bottom up Approach

In this approach, separated proteins or complex protein mixtures are

digested and the resultant peptides analyzed by MS in order to iden-

tify the native protein. The MS analysis is carried out either by pep-

tide mass fingerprinting or by sequence analysis using tandem mass

spectrometry. This is probably historical: 2D PAGE separated pro-

teins can only be extracted from the gel by digestion; high resolution,

nanoscale RPC of peptides supports high sensitivity MS analysis and

the performance and specification of mass spectrometers with

respect to peptides was higher than that for proteins. Fragmentation

of peptides, rather than whole proteins, has been more practical with

3 Mass Spectrometry272

mass spectrometers and mass accuracy greater for peptide analysis.

However, it does have its limitations. Acquiring complete or extensive

sequence coverage can be an issue, which can make protein charac-

terization problematic. Furthermore, the digestion process is largely

limited to trypsin, due to the fact that it locates basic residues to the

C-terminus of the peptide which is beneficial for fragmentation and

sensitivity, using CID tandem mass spectroscopy.

3.6.2

Top down Approach

This approach utilizes whole proteins and not digested peptides. This

approach is performed with mass spectrometers equipped with ETD

and ECD fragmentation. These techniques enable fragmentation of

large polypeptides and proteins.

Using this approach, accurate measurement of the molecular

weight and extensive sequence information can be acquired in one

analysis. Additionally, PTM information can be generated.

273

Functional Proteomics: Studies of Protein–Protein

Interactions

It is well known that the biological function cannot be linked to a sin-

gle protein. In fact proteins act in complexes and interact with other

proteins. Therefore the next step after expression proteomics is the

analysis of protein interactions. A few selected approaches are

described in brief in the following section. Many of these technolo-

gies, including SPR, are described in much more detail in the book

“Protein microarray technology” edited by Kambhamti (2003).

4.1

Non-immunological Methods

4.1.1

Separation of Intact Multi-protein Complexes

Because it provides a higher resolution than sucrose gradient centri-

fugation, the electrophoretic separation of intact protein complexes

with attached Coomassie brilliant blue dye in native polyacrylamide

gels has become well established in many proteomics laboratories.

Blue native PAGE had been developed by Schgger and von Jagow

(1991) for the separation of mitochondrial membrane proteins and

complexes in the mass range of 1,000 kDa to 10,000 kDa. Details on

the technique can be found in Section 1.2.3. In an alternative and

complementary approach multi-protein complexes are analysed by

electrospray ionization mass spectrometry (Lamond and Mann 1997).

4.1.2

Probing with Interaction Partners

“Far Western blotting” according to Burgess et al. (2000) is a non-anti-

body-based detection method for specific complex partners with a

purified bait protein after a native or a denaturating electrophoretic

separation.

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Kambhampti, D (ed) Protein

Microarray Technology. Wiley-

VCH, Weinheim (2003)

Schgger H, von Jagow G. Anal

Biochem. 199 (1991) 223–

231.

Lamond AI, Mann M. Trends

Cell Biol 7 (1997) 139–142.

Burgess R, Arthur TM, Pietz

BC. Methods Enzymol. 328

(2000) 141–157.

4 Functional Proteomics: Studies of Protein–Protein Interactions274

4.1.3

Surface Plasmon Resonance

Surface plasmon resonance (SPR) measures biomolecular binding

events in real time without the use of labels by detecting mass con-

centration changes on a sensor surface. SPR has become an estab-

lished technique for measuring biomolecular interaction, in particu-

lar protein interactions. SPR occurs when surface plasmon waves are

excited at a metal/liquid interface (the sensor surface). Light is directed

at, and reflected from, the side of the surface not in contact with sam-

ple. SPR causes a reduction in the reflected light intensity at a speci-

fic combination of angle and wavelength (generating a refractive index-

dependent SPR signal). Molecules binding to the sensor surface cause

changes in the refractive index close to the surface which are detected

as changes in the SPR signal. Specifically, in an SPR experiment a

reactant (ligand) is immobilized on a surface and its interaction with

a second component (the analyte) is measured. Effectively the asso-

ciation and dissociation of the ligand–analyte interaction is measured

(Fgerstam et al. 1992; see Figure 4.1). An SPR detector is capable of

measuring real time label-free biomolecular interactions.

Fig. 4.1: SPR principal of detection. SPR is used to measure

the change in refractive index as the analyte binds to the ligand

(from Biacore seminar).

A number of applications have been developed for the SPR analysis

of proteins with the objective of further characterizing protein inter-

actions. In particular, real time binding data is often essential to

understand the dynamic interactions between proteins and other bio-

molecules that drive and regulate biological processes. Specific ques-

tions may include what does the protein of interest bind with or to,

where does it bind, when does it bind and how does it bind? Interest-

ingly, studies with SPR can elucidate binding strength (affinity) speci-

Fgerstam LG, Frostell-Karlsson

, Karlsson R, Persson, B,

Rçnnberg, I. J Anal Chromato-

graphy 597 (1992) 397–410.

4.2 Antibody-based Techniques 275

ficity of interaction and the rates at which interactants bind and dis-

sociate or kinetics (on and off rates).

Using SPR, complex association and dissociation are detected in

real time and data are presented graphically in an interaction profile

known as a sensorgram, which is rich in information on dynamics of

molecular interactions (see Figure 4.2). A typical experiment takes

about 30 minutes to immobilize or capture a ligand protein, 3 min-

utes for sample injection and kinetic binding measurement, and 1

minute for regeneration. The latter two steps are repeated for a series

of analyses.

Fig. 4.2: Typical sensorgram of a label-free protein interaction analysis

with SPR. The signal height (measured in RU) is also prortional to the

mass of protein binding to the ligand.

Application areas in proteomics include the studies of protein com-

plexes, cell adhesion, signal transduction pathways, transcription,

regulation, and antibody recognition.

4.2

Antibody-based Techniques

4.2.1

Western Blotting and Dot Blots

The procedure of “Western blotting” is intensely described in Section

1.3. In many cases the efforts of electrophoretic separation of the

samples and transfer of the separated proteins onto the blotting

The major benefit of SPR tech-

nology is the information

gained from the kinetics of the

interaction.

However without pre-fractiona-

tion of the proteins cross-reac-

tions during the detection

procedure cannot completely

be excluded.

4 Functional Proteomics: Studies of Protein–Protein Interactions276

membrane is not necessary: The sample mixture possibly containing

the protein to be detected can also be applied directly on a blotting

membrane as a dot using dot blot or slot blot manifolds. The probing

for proteins is performed as described in Section 1.3.2. These dot

blots allow parallel screening of many samples.

4.2.2

Protein Microarrays

The concept described above, also called a “macroarray”, has mean-

while evolved into microarray technologies, using microscope slide

format (2575 mm). The basic technology for microarrys had been

developed for nucleic acid analysis in genomics and transcriptomics

studies. Similar methodologies are applied on expression profiling of

proteins in non-fractionated mixtures: Proteins are arrayed on mem-

branes or on microscope glass slides (protein chips) in very low

volumes in tiny spots using instruments similar to inkjet printers.

Either antibodies are printed on the carrier to capture specific ana-

lytes from body fluids, cell and tissue lysates; or – vice versa – the

sample lysates are immobilized on the carrier and antibodies are

used to detect specific sample components. Detection is usually per-

formed with a secondary antibody labeled with a fluorescent tag, like

cyanine dyes as used in the DIGE approach. In this way multiplexed

protein expression profiling can be carried out in a single assay; and

relative quantification is enabled. The spotting is fully automated by

employing pipetting robots, which apply the solutions from 384-well

plates. The images require high resolution multifluorescence scan-

ners.

Protein microarrays allow studying many biological events simulta-

neously with very small sample volumes, resulting in molecular fin-

gerprints on the protein level. Protein microarrays show still some

methodological limitation, however there is a huge potential for a

wide area of applications from high-throughput proteomics to clinical

diagnostics. Figure 4.3 shows a typical microarray image after scan-

ning with different wavelength fluorescence detection.

A critical step is the immobili-

zation of the proteins onto the

support. Random orientation of

the proteins can cause lossof

biological activity and/ or

recognition of its epitope by the

antibody.

Also their ease of use and their

possibility of automation

protein microarrays pledge to

be more suitable for diagnostic

applications than the classic

proteomic separation technolo-

gies.

4.2 Antibody-based Techniques 277

Fig. 4.3: Typical microarray image using Cy3 and Cy5

fluorescent labels after scanning with a high-resolution

version Typhoon multifluorescence scanner.

279

Part II:

Practical Manual of Proteome Analysis

Equipment, Consumables, Reagents 281

Step 1: Sample Preparation 287

Step 2: Fluorescence Difference Gel Electrophoresis 299

Step 3: Isoelectric Focusing 309

Step 4: SDS Polyacrylamide Gel Electrophoresis 323

Step 5: Scanning of Gels Containing Pre-labeled Proteins 357

Step 6: Staining of Gels 361

Step 7: Image Analysis and Evaluation of DIGE Gels 373

Step 8: Spot Excision 383

Step 9: Sample Destaining 387

Step 10: Protein Digestion 389

Step 11: Microscale Desalting and Concentrating of Sample 393

Step 12: Chemical Derivatization of the Peptide Digest 397

Step 13: MS Analysis 399

Step 14: Calibration of MALDI-ToF MS 403

Step 15: Preparing for a Database Search 407

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1