Sample preparation for peptide mapping--A pharmaceutical quality-control perspective.

In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the experimental procedure are therefore different than in the case of determination of an unknown protein structure. It is here recognized that a peptide-mapping method for quality control of proteins should be inert (not induce or revert modifications), general, robust, and allow a high sample throughput. The steps prior to the separation of the generated peptides are identified as crucial for meeting these demands. This includes denaturation, reduction, alkylation, buffer exchange, solubilization, and digestion. A critical review of the literature regarding these steps is presented. Relevant options in all steps are experimentally evaluated. Novel approaches are developed for many of the steps. The result is a sample preparation procedure that essentially meets the stated demands.

Complementary DNA and deduced amino acid sequences of procine alpha 1-microglobulin and bikunin.

Analysis of complementary DNA for porcine alpha 1-microglobulin and bikunin indicates that both proteins result from proteolytic processing of a common precursor similar to that found in man. Complete primary structures of these proteins are deduced from the nucleic acid sequence and partially confirmed by peptide sequencing.

Two out of the three kinds of subunits of inter-alpha-trypsin inhibitor are structurally related.

Inter-alpha-trypsin inhibitor is a 240-kDa plasma-protein complex of three different types of glycoproteins. Their stoichiometric relation in the complex is not yet known. One subunit results from proteolytic processing of a precursor protein composed of alpha 1-microglobulin and a double-headed Kunitz-type proteinase inhibitor protein. From this, only the inhibitor protein becomes part of the inter-alpha-trypsin inhibitor complex. Another subunit whose function is not yet understood is structurally unrelated to the first one as well as to other proteins of various data collections. Now we have obtained a cDNA clone coding for 837 amino acid residues of a precursor protein of the third subunit. Its primary structure is 40% identical to that of the completely known second-subunit precursor. Peptide sequences obtained from isolated inter-alpha-trypsin inhibitor represent a distinct part of only about two thirds of the predicted polypeptide precursor, suggesting that its maturation is very similar to that of the second subunit. Therefore, we conclude that the deduced primary structure covers about 98% of the mature third subunit.

Inter-alpha-trypsin inhibitor is a complex of three different protein species.

We found that, in contrast to earlier reports, inter-alpha-trypsin inhibitor of human serum is not a single-chain glycoprotein, but a protein complex. Three different protein components which are encoded on three independent mRNAs could be demonstrated by sequencing of cloned cDNA as well as of peptides obtained from purified inter-alpha-trypsin inhibitor. Accordingly, after fractionation of plasma by ammonium sulfate precipitation, ion-exchange chromatography, zinc-chelate chromatography, and SDS-polyacrylamide gel electrophoresis three proteins can be detected in Western blots using antibodies directed against inter-alpha-trypsin inhibitor.

Complementary DNA and derived amino acid sequence of the precursor of one of the three protein components of the inter-alpha-trypsin inhibitor complex.

Inter-alpha-trypsin inhibitor is composed of three distinct protein components. These protein components stem from independently encoded and proteolytically processed precursor proteins. Only the structure of the protein component responsible for the inhibitory activity has been established so far. We now present the complete amino acid sequence of the precursor of the second protein component derived from cloned cDNA. The precursor molecule includes both a signal peptide and a propeptide sequence and seems to be further processed prior to the assembly of the inter-alpha-trypsin inhibitor complex.

cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins.

Inter-alpha-trypsin inhibitor (ITI) is a serum protein of unknown function. Part of the molecule (formerly called HI30) is closely related to a tumor-derived protein acting as a growth factor for endothelial cells. We screened a human liver cDNA expression library with antibodies raised against human ITI and isolated several clones which could be divided into three groups according to their DNA sequences. The cDNA of the first group codes for a protein composed of alpha 1-microglobulin (alpha 1M) and urinary trypsin inhibitor (UTI) and is identical to that encoded by a clone originally found by screening a human liver cDNA library with oligonucleotides derived from amino-acid sequences of the two Kunitz-type domains of UTI. The proteins derived from the cDNA of the second and the third group of clones are distantly related to each other, but unrelated to the protein derived from group 1 clones. Partial amino-acid sequencing of ITI isolated from serum allowed the verification of large parts of the cDNA-derived amino-acid sequences. The results favour the view that ITI is not a single chain protein, but rather a very tight complex of several components or a mixture of such complexes.

[Primary structure of hemoglobin of the polar bear (Ursus maritimus, Carnivora) and the Asiatic black bear (Ursus tibetanus, Carnivora]. / Die Primärstruktur der Hämoglobine von Eisbär (Ursus maritimus, Carnivora) und Kragenbär (Ursus tibetanus, Carnivora).

The adult Polar and Asiatic Black Bear have one hemoglobin component each. The complete amino-acid sequences of their alpha- and beta-chains are presented. Their primary structures were determined by sequencing the tryptic and prolyl peptides. The alignment of these peptides was deduced from homology to human hemoglobin chains. The hemoglobin sequences of the two species proved to be identical. The evolutionary aspects of this result are discussed. A table of identical hemoglobin sequences from different species is given.