Dozens Of Peptides Research Articles

A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.

Peptides are widely used in biomaterials due to their easy of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for non-degradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. However, removing these constituents can be expensive, time consuming, and increases sample variability. Here we show that directly injecting samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration, and that hundreds of LC-MS runs can be done on a single column without a significantly diminish the ability to quantify the degradation of peptide libraries. We also show that column failure is evident when hydrophilic peptides fail to be retained on the column, and this can be easily identified using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.

Analysis of Proteins and Peptides of Highly Purified CD9+ and CD63+ Horse Milk Exosomes Isolated by Affinity Chromatography.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

Performing flow injection chromatography using a narrow open tubular column

Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3–5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity.

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea+1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95kDa) but also few dozens of peptides with molecular masses from 2 to 9.5kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.

Novel "omics" approach for study of low-abundance, low-molecular-weight components of a complex biological tissue: regional differences between chorionic and basal plates of the human placenta.

Tissue proteomics has relied heavily on two-dimensional gel electrophoresis, for protein separation and quantification, then single protein isolation, trypsin digestion, and mass spectrometric protein identification. Such methods are predominantly used for study of high-abundance, full-length proteins. Tissue peptidomics has recently been developed but is still used to study the most highly abundant species, often resulting in observation and identification of dozens of peptides only. Tissue lipidomics is likewise new, and reported studies are limited. We have developed an "omics" approach that enables over 7,000 low-molecular-weight, low-abundance species to be surveyed and have applied this to human placental tissue. Because the placenta is believed to be involved in complications of pregnancy, its proteomic evaluation is of substantial interest. In previous research on the placental proteome, abundant, high-molecular-weight proteins have been studied. Application of large-scale, global proteomics or peptidomics to the placenta have been limited, and would be challenging owing to the anatomic complexity and broad concentration range of proteins in this tissue. In our approach, involving protein depletion, capillary liquid chromatography, and tandem mass spectrometry, we attempted to identify molecular differences between two regions of the same placenta with only slightly different cellular composition. Our analysis revealed 16 species with statistically significant differences between the two regions. Tandem mass spectrometry enabled successful sequencing, or otherwise enabled chemical characterization, of twelve of these. The successful discovery and identification of regional differences between the expression of low-abundance, low-molecular weight biomolecules reveals the potential of our approach.

Peptide sequences converting polyglutamine into a prion in yeast.

Amyloids are ordered protein aggregates composed of cross-β sheet structures. Amyloids include prions, defined as infectious proteins, which are responsible for mammalian transmissible spongiform encephalopathies, and fungal prions. Although the conventional view is that typical amyloids are associated with nontransmissible mammalian neurodegenerative diseases such as Alzheimer's disease, increasing evidence suggests that the boundary between transmissible and nontransmissible amyloids is ambiguous. To clarify the mechanism underlying the difference in transmissibility, we investigated the dynamics and the properties of polyglutamine (polyQ) amyloids in yeast cells, in which the polyQ aggregates are not transmissible but can be converted into transmissible amyloids. We found that polyQ had an increased tendency to form aggregates compared to the yeast prion Sup35. In addition, we screened dozens of peptides that converted the nontransmissible polyQ to transmissible aggregates when they flanked the polyQ stretch, and also investigated their cellular dynamics aiming to understand the mechanism of transmission.

Y‐chromosome amelogenin isoform peptides found in mature dental enamel (918.1)

The dental enamel is the hardest and most calcified tissue in the human body, and most of its structure is unaltered during the time the teeth stay in mouth. The best known enamel specific structural proteins are amelogenin, ameloblastin and enamelin. The ability to recover proteins has enormous value in many fields where the study of proteins preserved from the past could give specific information on diet, life style, and even of biochemical evolution of the proteins themselves, since such information is not encoded in the DNA. So far, only X‐chromosome amelogenin peptides (AMELX) were recovered from mature enamel. Since amelogenin is encoded in the X and Y chromosomes in humans, peptides specific for the Y chromosome might exist in the mature enamel, but they have never been found. In this study we aimed at recovering peptides from male and female teeth, with the objectives of 1) finding Y‐encoded amelogenin peptides (if expressed at all, which is not known at the protein level) and 2) describing the aminoacid sequences of mature dental enamel peptides. Results showed that the Y‐specific amelgonenin (AMELY) sequence WYQSMIRPPY is found in male teeth and that with the enamel etch technique used here followed by nLC/MS/MS, dozens of peptides that are specific for enamel proteins were found (3 AMELX isoforms, 1 AMELY isoform, ameloblastin and enamelin).This report opens a myriad of possibilities for studying both present and long‐gone species, since dental enamel is a well preserved repository where such peptides can be detected.Grant Funding Source: Supported by FAPESP (Brazil)

Rapid Flow‐Based Peptide Synthesis

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.

Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis.

For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange is determined by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form.

Functional Continuum of Regulatory Peptides (RPs): Vector Model of RP-Effects Representation

During the past decades, bioactive (regulatory) peptides have been identified as the major players in the regulation of many important biological processes. Dozens of peptides have found their application as pharmaceutical agents, which further stimulated research in this field making it one of the most rapidly developing areas on the edge of biological science and medicine. However, the fast accumulation of enormous amounts of experimental data has revealed a great difficulty in their analysis and demanded the development of a systematic approach for generalization of the obtained information. We propose a new computer-based algorithm for studying biological activities of regulatory peptides and their groups based on their representation as vectors in n -dimensional functional space. Our method allows the rapid analysis of databases containing thousands of polyfunctional regulatory peptides with overlapping spectra of physiological activity. The described method permits to perform several types of correlations which, when applied to the large databases, could reveal new important information about the system of regulatory peptides. It can select the groups of peptides with similar physiological role (peptide constellations) and search for the optimal peptide combinations with predetermined spectrum of effects and minimal side effects for their further pharmacological application. It can also reveal the role of regulatory peptides in induction of chain physiological reactions.