Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.     

Ice nucleation in solutions and freeze-avoiding insects-homogeneous or heterogeneous?

This article challenges the common view that solutions and cold-hardy freeze-avoiding insects always freeze by heterogeneous nucleation. Data are presented to show that the nucleation temperatures of a variety of solutions and freeze-avoiding insects are a function of the water volume as described by the data previously published by Bigg in 1953. The article also points out that the relationships between melting point depression and depression of nucleation temperature are different for samples undergoing homogeneous nucleation and those undergoing heterogeneous nucleation. Aqueous solutions and freeze-avoiding insects display a relationship like that of homogeneously nucleated samples. It is also argued that the identity of the "impurities" assumed to cause heterogeneous nucleation in aqueous solutions and insects is obscure and that the "impurities" have features which make their existence rather unlikely.     

Genotypic Differences of Candida albicans and C. dubliniensis Isolates Related to Ethnic/Racial Differences within the Same Geographic Area

Candida albicans and C. dubliniensis genotype differences among Israeli ethnic groups were assessed. Isolates from Jews (51), Arabs (35) and Druze (25) were genotyped. The distributions among ethnic groups were not different, however they differed (p = 0.002) from global populations. Therefore, C. albicans and C. dubliniensis genotype distribution differences in Israel are related to changes in all ethnic groups.     

Localization of laminin alpha4-chain in developing and adult human tissues.

Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.     

CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F₁ populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F₁ populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F₁ populations. The mode of inheritance of the R10 allele could not be deduced as only very few F₁ hybrids bearing R10 were obtained. Linkage analysis in two F₁ populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.     

Chymotryptic-like hydrolysis of luliberin (LH-RF) by an adenohypophyseal enzyme of high molecular weight

An enzyme that hydrolyzes the fluorogenic chymotrypsin substrate glutaryl-Gly-Gly-Phe-β-naphthylamide has been partially purified from extracts of bovine anterior pituitaries. Like chymotrypsin, this enzyme hydrolyzes the neuropeptide Luliberin (LH-RF, <Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) at the carboxyl-side of Trp and Tyr, but it differs from the pancreatic protease by its high molecular weight, insensitivity towards OH-reactive agents and other enzymechemical parameters. It seems, however, to be identical to the “cation-sensitive neutral endopeptidase”. In the course of this study evidence has also been obtained that LH-RF is not degraded by the cystinyl-arylamidase.