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6 Cysteine and Methionine; Amino Acids Containing Sulfur

6 Cysteine and Methionine; Amino Acids Containing Sulfur

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Chapter 2

according to the Kyte and Doolittle scale1 is positive

and equals 1.9 and 2.5, respectively. Unlike cysteine,

the sulfur of methionine is not highly nucleophilic,

although it will react with some electrophilic centers.

It is generally not a participant in the covalent

chemistry that occurs in the active centers of enzymes.

The thiolate anion, which is formed after the ionization of cysteine in basic solutions, does not change the

character of this amino acid. Therefore, it is very

uncommon to find cysteine on the surface of a protein

even after ionization. The sulfur of methionine, as

with that of cysteine, is prone to oxidation; therefore,

oxidated methionine is added to database searches of

tandem mass spectra. The first step of oxidation,

yielding methionine sulfoxide, can be reversed by

standard thiol-containing reducing agents. The

second step yields methionine sulfone and is effectively irreversible. When oxidized, cysteine residues

can form disulfide bonds, strengthening protein

tertiary and quaternary structures. Additionally, many

metal-containing proteins use cysteines to hold their

metals in place, as the sulfhydryl side chain is a strong

metal binder. There are a few reasons why sulfur

atoms in amino acids do not affect the position of

those amino acids in proteins. One of the most

important aspects is the strong ability to create

disulfide bonds in comparison with the creation of the

weakest, noncovalent hydrogen bond with water.

However, the weakest ability to attract electrons (in

comparison to oxygen) results in a lack of hydrogen

bonds using sulfur atoms.

Cysteine stabilizes the tridimensional structure of

proteins, which is critical for extracellular proteins that

may be exposed to harsh conditions. Because proteins

containing multiple disulfide bridges are more resistant to thermal denaturation, they may maintain their

biological activity at more extreme conditions.

The existence of a disulfide bridge inside a protein

(intramolecular) and/or between different proteins

(intermolecular) makes it necessary to break those

bonds before proteomic analysis. The standard

approach is a two-step procedure that is almost

always applied to prepare proteins samples for proteomic analysis. In the first step, proteins are reduced

using dithiothreitol (C4H10O2S2) or mercaptoethanol,





although the latter agent is now used rather seldom.

In this step, disulfide bridges break, yielding free

sulfhydryl groups. In the following second step, free

sulfhydryl groups are alkylated to prevent reoxidation

and formation of bridges.

The chemical feature of cysteinedisotopic-coded

affinity tag (ICAT)dhas also been utilized in a gelfree MS-based technique for quantitative comparisons of up to two samples. This approach uses

a chemical reagent consisting of a thiol-reactive

group (labeling cysteines), linker and acid cleavable

biotin moiety (for affinity-based purification) as

presented in Figure 2.2.5,6 Quantification can be

performed using either carbon or hydrogen isotope

labeling. In case of carbon labeling, isotopic linkers

contain nine carbon isotopes 13C (heavy tag) and

nine carbon isotopes 12C (light tag). ICAT reagents

using labeled hydrogen atoms contain eight

hydrogen isotopes 2H (heavy tag) and eight hydrogen

isotopes 1H (light tag). Application of incorporated


C rather than 2H allows increase accuracy and

precision of quantification based on mass spectrometry using both electrospray ionization and

matrix-assisted laser desorption ionization techniques. A limitation of the ICAT technique is possible

quantification of only cysteine-containing proteins.

The biological importance of sulfur-containing

amino acids is multifold. Methionine is necessary for

the synthesis of proteins. It forms S-adenosyl-Lmethionine, which serves at a methyl donor in reactions, prevents fatty liver through transmethylation

and choline formation, and can lower toxic acetaldehyde levels in humans after alcohol ingestion. It also

plays an important role in preserving the structure of






















Thiol specific

reactive group

Figure 2.2 Schematic representation of ICAT reagent. X could be either hydrogen (light reagent)

or deuterium (heavy reagent). Eight 2H atoms could be used, making an 8-Da difference in a singly

charged or a 4-Da difference in a doubly charged fragment.

Chapter 2

cell membranes 7 and it has an important function for

some reactions involved in protein and DNA

synthesis.8 Cysteine is found in b-keratin, an important

component of skin, hair, and nails. A huge number of

disulfide bonds causing keratin can be very hard, such

as in nails or teeth, or flexible, such as in hair. The

smallest number of disulfide bonds creates soft keratin

in skin. The human body uses cysteine to produce the

antioxidant glutathione, as well as the amino acid

taurine. The body can also convert cysteine into

glucose for a source of energy. Cysteine also plays a role

in the communication between immune system cells.

2.7 Protein Identification and


High confidence protein identification and indepth characterization in one proteomic experiment

is the most favorable goal. Although new tools have

been developed during the last decade, the inherent

properties of proteins and peptides create limitations

of how much information can be obtained. For

example, using one enzyme for protein fragmentation

will generate peptides that can be too short or too

long. For a protein with high confidence, two or three

peptides are usually sufficient; however, it may not be

enough for characterization and/or identification of

specific regions of a protein. One example is histones.

These proteins contain multiple lysine residues in one

string and can be highly methylated and/or acetylated. It is an analytically challenging task to identify

the exact position of methylation or acetylation.

Therefore, protein characterization usually requires

more than one analytical approach, which will require

more biological material not always abundantly


2.8 StructureeFunction Relationship

and Its Significance in Systems

Biology Function

The major goal of proteomic profiling experiments is to get an insight into how the complex





biological system works; therefore, the most desirable outcome is new functional information. When

proteomics was born in the mid-1990s, everybody

was fascinated with the ability to identify (catalog)

tens, hundreds, and then thousands of proteins

in one analytical experiment. This did not last long,

as we realized that answers are in relative quantitation rather than the presence or absence of

a particular protein. At this point we hit yet another

wall, which was post-translational modifications,

which increased the complexity of proteomic

experiments by at least two orders of magnitude.

New experimental approaches have been proposed

and collectively great progress has been made in

accumulating huge amounts of data. Although

significant steps in the biological interpretation of

such massive data have been made, our knowledge

about how biological systems function is growing at

a disproportionally low rate. Two hurdles in progress here are correlation of protein structure and

function and protein localization and function. The

latter phenomenon is also called protein moonlighting. This brings us to question what a protein

structure represents in defining its biological function and further on how the structure of a protein

defines its physiological function.

What if we assume that similar sequences of

proteins represent similar functions, whereas

different sequences are responsible for different

functions? We will certainly find many examples to

support such assumptions. Let us consider transmembrane domains of receptors that are hydrophobic and have a helical structure to be

accommodated by a hydrophobic environment of

a lipid bilayer. Furthermore, integrins a 1, 2, and 4

have single-pass transmembrane helical domains

that all play the same function: anchoring these

proteins into the cell membrane. They are all close to

the C-terminal end of the polypeptide chain;

however, all of them have a different primary structure (Figure 2.3).

As we know, integrins are responsible for transmitting signals related to numerous functions and

are part of a/b heterodimers.

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6 Cysteine and Methionine; Amino Acids Containing Sulfur

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