Lecture 6 Summary

Proteins: Polypeptide diversity, protein purification and analysis. Chromatography and electrophoresis. (4: 91-103).

"Classical" protein sequencing by enzymatic and chemical methods. Protein cleavage and separation of peptide fragments. The Edman degradation is used to chemically sequence peptides (4: 104-110).

Mass spectrometry of proteins and peptides: The electrospray ionization (ESI) method has facilitated analysis of biological macromolecules by mass spectrometry. Molecular masses of peptides or proteins can be determined and short polypeptides can be directly sequenced. For the latter, tandem mass spectrometry (or MS/MS), is employed. Peptide sequences are assembled into complete protein sequences, and researchers have submitted thousands of such sequences and accompanying annotations to publically-accessible databases. Currently, collaborative efforts are directed toward establishment of UniProt as a unified worldwide protein sequence and function database (4: 110-113).

Protein evolution (4: 114-120).

Protein purification and characterization (outline)

Analysis of individual proteins largely takes place under in vitro, meaning "test tube" conditions outside of the cell. Protein purification methods are used to isolate a protein as a relatively pure sample under conditions where its activity can be preserved. Many purified protein samples are most effectively prepared by heterologous expression, or overexpression in a non-native host cell. Laboratory strains of the common enterobacterium E. coli are routinely employed for this purpose.

Steps in protein purification

Source of protein: "native" or heterologous expression
Importance of an assay
Disruption of tissue or cell suspension, producing homogenate
Fractionation of homogenate by centrifugation
"Salting out"
Dialysis
Chromatographic separation. types of chromatography: (i) ion exchange (ii) gel filtration (iii) affinity

Electrophoretic analysis of proteins

• PAGE: polyacrylamide gel electrophoresis
• Denaturing PAGE: SDS-PAGE - separation on the basis of size
• Isoelectric focusing: separation of proteins on the basis of pI (isoelectric point)
• 2D-gels: combines isoelectric focusing and SDS-PAGE

pI, pH, acidic & basic proteins

• The pI of a protein is the pH at which the net charge on the molecule is zero
• If pH > pI, then the protein is negatively charged (acidic proteins have pI < 7)
• If pH < pI, the protein is positively charged (basic proteins have pI > 7)

Techniques for chemical and enzymatic cleavage of proteins

Chemical cleavage: Cyanogen bromide (CNBr) cleaves after Met residues; hydroxylamine cleaves Asn-Gly bonds.

Enzymatic cleavage: Relies on proteases with well-defined specificities. Examples: trypsin (after Arg, Lys residues), clostripain (after Arg residues), thrombin, chymotrypsin, carboxypeptidase A.

Immunological-reagent methods

Western blotting

Immunofluorescence

Peptide methods

Peptide sequencing

Edman degradation: proceeds from N terminus. Peptide mapping

Peptide synthesis

Synthetic peptides are useful reagents. Solid-phase peptide synthesis.
Synthesis is performed by automated solid-phase methods; proceeds in C to N direction

Biophysical methods

Sedimentation analysis (Ultracentrifugation); Mass spectrometry; Circular dichroism.

A MALDI-TOF example (Ref.4). An electrospray and protein mass determination example (VVP3e). Advanced special topic: Peptide sequencing by MS

Proteomics

The proteome can be defined as "the functional representation of the genome” (Ref. 4, p.66). From this it would follow that proteomics is the study of the functional consequences of various and varying patterns of gene expression. Building on the reductionist study of individual proteins in vitro, the characterization of how proteins interact with their environment in vivo has progressed rapidly. We now possess an appreciation for the dependence of protein function upon context. Structures are available for many protein complexes ranging from simple binary protein-protein or protein-nucleic acid complexes all the way up to structures of viruses and the ribosome. Such "supramolecular" assemblies will determine the context-dependent functional roles of the components. One example concerns the accessory proteins that interact, together with the enzyme RNA polymerase, to form a DNA-bound complex, a supramolecular structure that is capable of initiating and completing transcription of a gene. The particular cast of characters assembled in this complex - some regulars, others more variable - will determine whether a gene is being transcribed, and whether this gene expression is at basal or enhanced levels. At a cellular level, the patterns of gene expression proteomics seeks to understand are correlated with metabolic resources and status, developmental or cell-cycle stage, state of differentiation, and environmental cues. Other variables for which proteomics must account are the existence of protein isoforms and the effects of post-translational modifications. The function or lifetime of a protein can be radically altered by specific cleavage of the polypeptide chain itself or attachment of various moieties such as phosphates, sugars - even other proteins. Isoforms of proteins can arise via expression from separate genetic loci or alternatively-spliced mRNAs.

The human genome project and the sequencing of many other genomes has generated considerable amounts of information, necessitating development and application of a computational infrastructure for storage, retrieval, annotation, and analysis of all this information. Computational sciences will be central to development of the science of proteomics, if only because of the astronomical expansion of proteomic information in relation to that characterizing the cognate genome. Furthermore, computational theories and methods treating problems such as pattern recognition, networks, and emergent properties of complex systems can applied to biological and biomedical questions. For example, physical interactions between proteins and their functional cooperation can be modeled as networks. Such "systems"-level approaches may provide crucial insights on diseases and their treatment.

Examples of posttranslational modifications

(1) Hydroxylation - e.g. hydroxyproline in collagen (Vitamin C-dependent).
(2) Carboxylation - e.g. carboxyglutamate in prothrombin (Vitamin K dependent)
(3) Glycosylation - very common in higher eukaryotes. O-linked sugars are attached to Ser and Thr, N-linked sugars are attached to amide nitrogen of Asn side chain.
(4) Phosphorylation - a major regulatory mechanism in signal transduction. Most commonly involves transfer of phosphate group from ATP to the oxygen of Ser, Thr, or Tyr side chains.

Learning objectives

• Define the term proteome and describe the concepts underlying proteomics.
• Describe the most common post-translational modifications.
• Describe several key analytical methods applicable to proteomics.
• Describe how the charge of a protein varies with pH.
• For each of the following techniques, describe the principles upon which it is based, the type of information obtained through its use, and any significant limitations:
  • Chemical and enzymatic cleavage of polypeptide chains
  • Electrophoretic techniques:
    • SDS-PAGE
    • Isoelectric focusing
    • Two-dimensional gel electrophoresis
    • Western blotting
  • Chromatography and chromatographic techniques:
    • size-exclusion
    • ion-exchange
    • affinity
    • hydrophobic interaction
• For each of the following techniques, describe the principles upon which it is based, the type of information obtained through its use, and any significant limitations:
  • Automated peptide sequencing (Edman degradation)
  • Peptide mapping and direct protein sequence determination.
  • Indirect protein sequence determination
  • Automated solid-phase peptide synthesis.

Page updated 08-04-2010

References:

1. Creighton, TE. Proteins: Structure and Molecular Properties (2nd ed, 1993. Freeman)
2. Branden, Carl & Tooze, John Introduction to Protein Structure (1st ed. 1991, Garland Publishing)
3. Siuzdak G. Mass Spectrometry for Biotechnology (1996, Academic Press)
4. Berg JM, Tymoczko JL, Stryer L. Biochemistry (6th edition) (Freeman).