Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

Molecular structures and sequences are generally more revealing of evolutionary relationships than are classical phenotypes (particularly so among microorganisms). Consequently, the basis for the definition of taxa has progressively shifted from the organismal to the cellular to the molecular level. Molecular comparisons show that life on this planet divides into three primary groupings, commonly known as the eubacteria, the archaebacteria, and the eukaryotes. The three are very dissimilar, the differences that separate them being of a more profound nature than the differences that separate typical kingdoms, such as animals and plants. Unfortunately, neither of the conventionally accepted views of the natural relationships among living systems--i.e., the five-kingdom taxonomy or the eukaryote-prokaryote dichotomy--reflects this primary tripartite division of the living world. To remedy this situation we propose that a formal system of organisms be established in which above the level of kingdom there exists a new taxon called a "domain." Life on this planet would then be seen as comprising three domains, the Bacteria, the Archaea, and the Eucarya, each containing two or more kingdoms. (The Eucarya, for example, contain Animalia, Plantae, Fungi, and a number of others yet to be defined). Although taxonomic structure within the Bacteria and Eucarya is not treated herein, Archaea is formally subdivided into the two kingdoms Euryarchaeota (encompassing the methanogens and their phenotypically diverse relatives) and Crenarchaeota (comprising the relatively tight clustering of extremely thermophilic archaebacteria, whose general phenotype appears to resemble most the ancestral phenotype of the Archaea.

https://www.pnas.org/content/pnas/87/12/4576.full.pdf

Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.

BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

“Bioinformatics” 특집을 내면서

NF-κB (nuclear factor-κB) is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-κ...

Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity.

Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, conside...

The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction

An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP) and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues) or CGRPs (37 amino acids) and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

https://pure.mpg.de/pubman/item/item_2262606_2/component/file_2262608/1-s2.0-S2212968515300106-main.pdf

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

Separation sciences in the era of proteomics

Amyloid deposits today represent 37 different amyloid diseases [1,2]. The many classes of amyloidogenic proteins are exemplified as immunoglobulins, apo-lipoproteins, hormones and many other unrela...

Cathepsin K as a novel amyloid fibril protein in humans.

/pdf/the-structural-error-and-its-relation-to-the-malfunction-in-364xiynj7y.pdf

Friedrich Lottspeich

Papers

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

Separation sciences in the era of proteomics

Cathepsin K as a novel amyloid fibril protein in humans.

The Structural Error And Its Relation To The Malfunction In Some Abnormal Fibrinogens

Limitations of high-speed reversed-phase high-performance liquid chromatography observed with integral membrane proteins