EDUCACIÓN Y FORMACIÓN AMBIENTAL: MARCO INTERNACIONAL

1.4.1 Molecular mass determination

The molecular mass of a protein is an important parameter in its biochemical characterisation. SDS-PAGE is a universal technique for determining the molecular mass o f a protein, with an accuracy ranging from a few percent for a typical unglycosylated globular protein to about 30% for a heavily glycosylated protein (Nguyen, 1995). This makes the accurate determination of molecular mass by this method unreliable. With the recent advances in ionisation techniques it is obvious why mass spectrometry of a protein has become such a useful tool for protein molecular mass measurement. Techniques such as MALDI and ESI allow the determination o f molecular weight o f a protein at the low pmol level for masses in excess o f 100,000 Da and with much greater precision.

ESI-MS can determine the mass of the protein with an accuracy exceeding 0.1%, which surpasses the best results obtained by SDS-PAGE. Although ESI can measure molecular masses above 150,000 Da (Siuzdak, 1994), the analyses become complicated to interpret as the mass exceeds this figure. Recent advantages in MALDI TOF instruments fitted with time-lag focusing have brought molecular mass determination t o nearly comparable to that o f ESI. H owever, although continuous flow MALDI analysis is being developed, it is not yet commercially available. Currently only ESI-MS methods for molecular mass determination are automated, with a high throughput o f samples and the determination o f the mass o f a protein in minutes.

1.4.2 Peptide mapping

As the genomes of various organisms are sequenced, the ever increasing number of databases containing protein sequence information has become a valuable aid to

C h apter 1.___________________________________Introduction

identification of proteins by mass spectrometry. A key component o f strategies for defining molecular pathways is the development of a rapid and sensitive method for generating an 'address’ with which a protein can be found in a database. Mass spectrometry has revolutionised this area.

Traditionally, short stretches of amino acid sequences were used to search databases to locate protein or gene sequences, or to identify proteins o f similar sequence. The short amino acid sequence constituted a unique address for a protein and generally led to successful searches. The amino acid sequence information was usually obtained by N-terminal sequencing using Edman sequencers, a slow time-consuming process. In many c ases the N-terminal o f the protein was found to be blocked or modified, either naturally or artefactually, preventing the initial coupling reaction o f the Edman degradation and prematurely terminating the sequencing.

In 1981, peptide mapping techniques using mass spectrometry were introduced by Henzel and colleagues (1993) to determine the masses o f peptides resulting from the proteolytic and chemical cleavage of a protein, to verify gene sequences. It was quickly found that by combining molecular weight determination o f peptides obtained from a proteolytic digest, with the information in a database (Geneva, Switzerland, http://www.expasy.ch), a powerful and rapid approach for protein identification was possible. B y comparing e xperimental p eptide m asses from p roteins a fler c hemical

cleavage or proteolytic digest, with the predicted

theoretical masses (Protein Prospector, University o f San Francisco

(http://falcon.ludwig.ucl.ac.Uk/mshome3.2.htm). it has been shown that a peptide mass map produces a highly informative fingerprint (Clauser et a l, 1999).

This technique is not only a rapid and sensitive method for the identification of known proteins but has also been useful for identifying frame shift /deletion /insertion mutations, co- and post-translational and chemical modifications and for confirming the gene sequence. This is achieved by detecting changes in mass resulting from modifications to the peptide fragment.

C hapter 1.___________________________________Introduction

1.4.3 Post-translational modifications

1.4.3.1 Disulphide bond assignment

Many proteins contain disulphide bonds between sulphur atoms o f cysteine residues. Characterisation of these bonds is very important, especially in recombinant protein technology, since the formation o f disulphide bonds and protein folding are not under direct genetic control. Disulphide bridges play a key role in the determination o f the tertiary structure o f a protein. The native, correctly folded state o f a protein is stabilised by these disulphide bonds and is important in the 3D-structure o f a protein. Therefore a protein with the correct amino acid sequence but incorrectly formed disulphide bonds may not assume its active conformation. It is important to note that proteins with the wrong conformation can be distinct antigenically from the natural protein. Thus, it is important not only to establish the total cysteine /cystine content of a protein but also to determine which cysteines residues are involved in disulphide bridges.

Total cysteine content o f a protein or peptide can be determined quickly and accurately using mass spectrometry. The current technique involves the complete reduction o f all cysteines involved in disulphide bonds within the protein (using DTT or DTE) and the determination o f the molecular mass by mass spectrometry. The reduced protein is derivatised with iodoacetamide, which acetylates the R-S-H group in a cysteine residue and the molecular mass determined by mass spectrometry. For every derivatised cysteine residue in the protein there will be an increment in the mass to charge ratio of 57, corresponding to the mass o f the acetamide-derivatised cysteine residue. By subtracting the mass o f the reduced protein from the mass o f the derivatised protein and then dividing by the mass o f acetamido group (57 m/z) (Nguyen et al, 1995), it is possible to ascertain the number o f cysteines present in the protein. Which cysteine bonds are involved in disulphide bridges, can be deduced by the derivatisation o f the cysteine sulphur groups with iodoacetamide, without the prior reduction of the disulphide bridges using DTT or DTE. This way only the cysteines that are not involved in disulphide bridges are derivatised. The number of cysteine

C h apter 1.___________________________________Introduction

residues involved in disulphide bridges can be determined by subtracting the mass of the derivatised but not reduced protein, from the mass o f the fully reduced and derivatised molecule, and then dividing by the mass of the acetamide group.

1.4.3.2 Phosphorylation

Another common post-translational modification o f proteins is phosphorylation of serine and threonine residues. Phosphorylation o f a peptide or protein can be readily identified in mass mapping studies by a characteristic mass gain o f 80 Da due to the presence of a phosphate group on the peptide fragment after cleavage or digestion. A mass discrepancy o f 80 Da, from the expected peptide mass, would indicate the phosphorylation o f the peptide (Zhang et al, 1995). The peptide can then be analysed further by using additional digests to find the phosphorylation site. Alternatively the peptide can be sequenced to identify a mass increase o f 80 Da above the expected mass o f an amino acid. A rapid way of determining whether or not a peptide is phosphorylated is to use tandem-MS to perform a parent ion scan o f 80 m/z. In this way only phosphorylated peptides within a mixture are detected.

C h apter 1.___________________________________Introduction

1.4.3.3 Glycosylation

Glycosylation is the most common post-translational covalent modification observed in eukaryotic proteins. It can have a profound influence on the properties o f the glycoprotein. These include biological activity, immunogenicity, clearance rate, solubility, thermal stability and proteolytic resistance (see section 1.6 for an in depth description o f glycosylation). The study o f glycosylation has become an important field o f research, especially in understanding human disease and in the therapeutic use of recombinant protein technology, where rapid and accurate monitoring of glycosylation patterns is vital in quality control o f the product. Recombinant glycoproteins generally exist as a set o f glycosylated variants exhibiting heterogeneity with respect to both the proportion of potential glycosylation sites that are occupied i.e. macroheterogeneity, and the oligosaccharide structures present at each glycosylation site i.e. microheterogeneity (Varki, 1993).

The precise analysis of site-specific glycosylation and the structural analysis of carbohydrates are still major challenges for conventional analytical methods. A number o f techniques for structural analysis of glycoproteins are available including, lectin-binding, various forms of chromatography, flurophore-assisted carbohydrate electrophoresis (Mechref and Novotny, 2002) and the use o f highly specific endo- and exoglycosidase enzyme assays (Guile et al, 1996). However, these methods can be slow, laborious or require relatively large amounts o f sample. Hence, mass spectrometry is becoming a major technique for the analysis of protein glycosylation. It is capable of providing complete structural analysis of the branched nature of the glycans, linkage of one monosaccharide unit to its neighbour and the number of possible structural sugar isomers that may be involved. More significantly mass spectrometry requires s ubstantially 1 ess glycoprotein fo r analysis than that o f other procedures. A flow chart i llustrating how mass spectrometry m a y b e used in the analysis o f glycoproteins and carbohydrates is shown in Figure 1.09.

One o f the quickest and simplest ways of gaining a significant amount o f information on the carbohydrate content of a protein can be obtained from an

Figure 1.09

Glycoprotein analysis