Amino acid sequence, determination

Scheme 1 The Expected Fragmentation Pattern of Protonated Peptide Ions and the Nomenclature of the Amino Acid Sequence Fragment Ions for Determining Amino Acid Sequences...

Determining Amino Acid Sequence 149 J- The Protein Data Bank, Three-Dimensional... 

The information that determines amino-acid sequence in a protein to be synthesized is contained in the DNA of a cell nucleus as a particular sequence of nucleotides derived from adenine, guanine, thymine, and cytosine. For each particular amino acid there is a sequence of three nucleotides called a codon. 

Sequence repeats in proteins make use of structural and functional motifs that nature has found to work well in vivo. Large numbers of these motifs have now been characterized, and any newly determined amino acid sequence is generally run through computer databases to assess the number and type of repeats present, their possible conformations, and their likely functions in vivo. The characteristics of the sequence motifs... 

The sequence of amino acids in a protein is of critical importance in determining the function of that protein. Therefore, considerable effort has been devoted to the development of methods to determine amino acid sequences. The process usually begins with the determination of the relative numbers of each amino acid that are present in the protein. To accomplish this, a sample is completely hydrolyzed to its individual amino acid components by treatment with 6 M HC1 at 100 to 120°C for... 

Determining amino acid sequences of proteins by classic procedures is a tedious process. In addition, proteins are often insoluble (e.g., membrane proteins) or cannot be easily purified. However, if the protein s gene, mRNA, or cDNA of the mRNA are available, the amino acid sequence of the protein can be determined from nucleotide sequences rapidly and unambiguously using the universal genetic code. In this code, each amino acid in correlated with one or more nucleotide triplets in mRNA (see Chapter 12). mRNA is "transcribed" from DNA and can be used to synthesize complementary (cDNA) via an enzyme called reverse transcriptase. In many cases it is easier to isolate the mRNA or cDNA of a protein than the protein itself. 

An adage of biochemistry is, Never waste pure thoughts on an impure protein. Starting from pure proteins, we can determine amino acid sequences and evolutionary relationships between proteins in diverse organisms and we can investigate a protein s biochemical function. Moreover, crystals of the protein may be grown from pure protein, and from such crystals we can obtain x-ray data that will provide us with a picture of the protein s tertiary structure—the actual functional unit. 

Mass spectrometry has been applied to the structure elucidation of peptides and proteins for some time. Depending on the problem, it is used as both a primary and a complementary technique and although many difficulties still remain it is now an established means of determining amino acid sequences. As instrumental methods and new derivatives have developed, the different approaches employed, with particular combinations of chemical pretreatment and sample introduction to the MS, have been dictated by the size and type of the peptide and the information required. 

Both methods require complex mass spectrometers that can perform both MS and MS/MS measurements on the tryptic peptides. MS/MS involves the ability to further fragment proteins within the mass spectrometer along the peptide backbone to determine amino acid sequence information. The proteins are identified by comparing... 

When the methodology of 2D gel for separation of proteins became available in the mid-1970s, the proteins separated by 2D gel were routinely subjected to Edman degradation to determine the amino acid sequence, because this was the only way to determine amino acid sequence of a protein at that time. However, there are two problems with this approach in proteomics First, many proteins possess N-terminal proteins that are blocked and therefore cannot react with the phenylisothiocynate required for Edman degradation. Second, Edman degradation can determine the amino acid sequence of one protein at a time, which is contrary to the objectives of proteomics that aim at gaining the information of several proteins at the same time. 

ESI is frequently applied in the various stages of the characterization of peptides and proteins molecular mass determination, amino acid sequencing, determination of nature and position of chemical and posttranslational modifications of proteins, investigation in protein tertiary and quaternary conformations, and the study of noncovalent associates. In most cases, no online separation is applied, but the sample solution is introduced directly via the ESI or... 

Another important reason for determining amino acid sequences is that they provide a chemical tool for studying our evolutionary history. Proteins resemble one another in amino acid sequence if they have a common evolutionary ancestry. Let s look at a specific example. 

The technical problems in determining amino acid sequences in H chains are quite different from those encountered with L chains. Because of the large size of an H chain, complete sequence analysis is a formidable problem. Also, the N-terminus in many H chains is blocked as a result the number of known N-terminal sequences of H chains is relatively small by comparison with L chains. An exception is the Vnni... 

Additionally, the nitrocellulose surface provides an opportunity to carry out enzymatic and chemical reactions directly on the sample foil. These are discussed below, and in more detail later in Chapter 10, and include carboxy and amino-peptidase reactions that can be used to produce peptide ladder mixtures for determining amino acid sequence or the location of post-translational modifications. Following the on-foil reaction, the enzyme can be washed off prior to PDMS analysis of the resulting products. 

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