De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging

Proteomic studies require efficient, robust, and practical methods of characterizing proteins present in biological samples. Here we describe an integrated strategy for systematic proteome analysis based on differential guanidination of C-terminal lysine residues on tryptic peptides followed by capillary liquid chromatography-electrospray tandem mass spectrometry. The approach, termed mass-coded abundance tagging (MCAT), facilitates the automated, large-scale, and comprehensive de novo determination of peptide sequence and relative quantitation of proteins in biological samples in a single analysis. MCAT offers marked advantages as compared with previously described methods and is simple, economic, and effective when applied to complex proteomic mixtures. MCAT is used to identify proteins, including polymorphic variants, from complex mixtures and measure variation in protein levels from diverse cell types.     

Definition and characterization of a "trypsinosome" from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures

Although HPLC-ESI-MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC-ESI-MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC-MS/MS system on which data are being generated.     

An integrated, directed mass spectrometric approach for in-depth characterization of complex peptide mixtures

LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mixtures. For very complex samples such as complete proteomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abundance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detectable peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. The selected features or a subset thereof are subsequently sequenced in sequential, non-redundant directed LC-MS/MS experiments, and the MS/MS data are mapped back to the original LC-MS feature map in a fully automated manner. The strategy, implemented on an LTQ-FT MS platform, allowed the specific sequencing of 2,000 features per analysis and enabled the identification of more than 1,600 phosphorylation sites using a single reversed phase separation dimension without the need for time-consuming prefractionation steps. Compared with conventional DDA LC-MS/MS experiments, a substantially higher number of peptides could be identified from a sample, and this increase was more pronounced for low intensity precursor ions.     

Adjustment to the light environment in small-statured forbs as a strategy for complementary resource use in mixtures of grassland species

Background and Aims

The biological mechanisms of niche complementarity allowing for a stable coexistence of a large number of species in a plant community are still poorly understood. This study investigated how small-statured forbs use environmental niches in light and CO₂ to explain their persistence in diverse temperate grasslands.

Methods

Light and CO₂ profiles and the corresponding leaf characteristics of seven small-statured forbs were measured in monocultures and a multi-species mixture within a biodiversity experiment (Jena Experiment) to assess their adjustment to growth conditions in the canopy.

Key Results

Environmental conditions near the ground varied throughout the season with a substantial CO₂ enrichment (>70 µmol mol⁻¹ at 2 cm, >20 µmol mol⁻¹ at 10 cm above soil surface) and a decrease in light transmittance (to <5 % deep in the canopy) with large standing biomass (>500 g d. wt m⁻²) in the multi-species assemblage. Leaf morphology, biochemistry and physiology of small-statured forbs adjusted to low light in the mixture compared with the monocultures. However, the net carbon assimilation balance during the period of low light only compensated the costs of maintenance respiration, while CO₂ enrichment near the ground did not allow for additional carbon gain. Close correlations of leaf mass per area with changes in light availability suggested that small-statured forbs are capable of adjusting to exploit seasonal niches with better light supply for growth and to maintain the carbon metabolism for survival if light transmittance is substantially reduced in multi-species assemblages.

Conclusions

This study shows that adjustment to a highly dynamic light environment is most important for spatial and seasonal niche separation of small-statured forb species in regularly mown, species-rich grasslands. The utilization of short-period CO₂ enrichment developing in dense vegetation close to the ground hardly improves their carbon balance and contributes little to species segregation along environmental niche axes.     

Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex

Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.     

RAPD technique is a useful tool to distinguish Penicillium species.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic diversity among 13 soil Penicillium strains originating from widely dispersed areas. Twenty one of the 34 synthetic random primers were found to identify polymorphism in amplification products. The results show a high level of diversity of RAPD markers among the strains. All the strains could be identified by their characteristic amplification profile, using selected random primers. This suggests that RAPD analysis is a useful and reliable assay for characterizing the species of Penicillium genus.     

Inactivation of C5a anaphylatoxin by a peptide that is complementary to a region of C5a

PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37-53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all. C5a was inactivated by concentrations of 7 nM or more of Pep-A, and this concentration of Pep-A inhibited induction of intracellular Ca(2+) influx in neutrophils. Patch clamp electrophysiology experiments also showed the effectiveness of Pep-A in C5aR-expressing neuroblastoma cells. Furthermore, Pep-A administration prevented rats from C5a-mediated rapid lethal shock induced by an Ab to a membrane inhibitor of complement after LPS sensitization.     

Development of an immunological technique for identifying multiple predator–prey interactions in a complex arthropod assemblage

A simplified but highly effective approach for the post‐mortem evaluation of predation on several targeted members of an arthropod assemblage that does not require the development of pest‐specific enzyme‐linked immunosorbent assay (ELISA) (e.g. pest‐specific monoclonal antibodies) or PCR assays (DNA primers) is described. Laboratory feeding studies were conducted to determine if predation events could be detected from predators that consumed prey marked with foreign protein. I determined that large and small rabbit immunoglobulin G (IgG)‐marked prey can be detected by a rabbit‐IgG‐specific ELISA in the guts of chewing and piercing–sucking type predators. I then conducted multifaceted inclusion and exclusion field cage studies to qualify the degree of interguild and intraguild predation occurring among a complex arthropod assemblage during four separate light phase treatments. The field cages contained an arthropod assemblage consisting of 11 or 12 species of predaceous arthropods and three pest species. The three pests introduced into the cages included third instar Trichoplusia ni marked with rabbit IgG, third instar Lygus hesperus marked with chicken IgG and Pectinophora gossypiella sentinel egg masses. The inclusion cages allowed foraging fire ants, Solenopis xyloni, to freely enter the cages while the exclusion cages contained barriers that prevented ant entry. The results obtained using the conventional inclusion/exclusion field cage methodology revealed that there was substantial interguild and intraguild predation occurring on the majority of the arthropods in the assemblage, particularly in those cages that included ants. I then precisely identified which predators in the assemblage were feeding on the three targeted pests by conducting three post‐mortem gut content analyses on each individual predator (1503 individuals) in the assemblage. Specifically, P. gossypiella egg predation events were detected using an established P. gossypiella‐egg‐specific ELISA, and third instar T. ni and L. hesperus predation events were detected using rabbit‐IgG‐specific and chicken‐IgG‐specific ELISAs, respectively. Generally, the gut ELISAs revealed that Collops vittatus, Spanagonicus albofasciatus and Geocoris punctipes readily preyed on P. gossypiella eggs; Nabis alternatus, Zelus renardii and spiders (primarily Misumenops celer) readily preyed on marked L. hesperus nymphs, and spiders, S. albofasciatus and N. alternatus readily preyed on T. ni larvae. Furthermore, the cage methods and the post‐mortem predator gut ELISAs revealed very few distinctive patterns of predation with regard to the light cycle the assemblage was exposed to.     

Active-site peptide "fingerprinting" of glycosidases in complex mixtures by mass spectrometry. Discovery of a novel retaining beta-1,4-glycanase in Cellulomonas fimi

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.     

Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III

The ubiquinone-binding protein (QP-C) is a nuclear-encoded component of ubiquinol-cytochrome c oxidoreductase in the mitochondrial respiratory chain and plays an important role in electron transfer as a ubiquinone-QP-C complex. We obtained a partial cDNA for rat liver QP-C by screening a lambda gt11 rat liver cDNA library using antiserum directed against bovine heart QP-C. Using this cDNA as a probe, a cDNA clone was isolated from a human fibroblast cDNA library by colony hybridization. The total length of the cloned cDNA was 518 base pairs with an open reading frame of 333 base pairs. The 111-amino acid sequence deduced from the nucleotide sequence of the cDNA is 85% homologous to that of bovine QP-C and contains only a single additional amino-terminal methionine. This implies that the human QP-C is synthesized without a presequence which is required for import of most nuclear-encoded mitochondrial proteins into mitochondria.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Developments in mass spectrometry for the analysis of complex protein mixtures.

State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein-peptide separation, mass spectrometry and data analysis. While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms. The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybrid mass spectrometers and application of emerging types of tandem mass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Preparative peptide isoelectric focusing as a tool for improving the identification of lysine-acetylated peptides from complex mixtures

Protein sequence database searching of tandem mass spectrometry data is commonly employed to identify post-translational modifications (PTMs) to peptides in global proteomic studies. In these studies, the accurate identification of these modified peptides relies on strategies to ensure high-confidence results from sequence database searching in which differential mass shift parameters are employed to identify PTMs to specific amino acids. Using lysine acetylation as an example PTM, we have observed that the inclusion of differential modification information in sequence database searching dramatically increases the potential for false-positive sequence matches to modified peptides, making the confident identification of true sequence matches difficult. In a proof-of-principle study of whole cell yeast lysates, we demonstrate the combination of preparative isoelectric focusing using free-flow electrophoresis, and an adjusted peptide isoelectric point prediction algorithm, as an effective means to increase the confidence of lysine-acetylated peptide identification. These results demonstrate the potential utility of this general strategy for improving the identification of PTMs which cause a shift to the intrinsic isoelectric point of peptides.     

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.     

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.     

A technique for identifying the roots of different species in mixed samples using nuclear ribosomal DNA

Abstract. Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots? Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multi‐species samples. Results: The technique successfully identified species present in multi‐species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each species. Conclusions: This method is the first to identify the species composition of mixed root samples. It should be applicable to most plant species because the ITS region is flanked by universal primers and most species have unique ITS sequences. The ability to determine species‐specific rooting distributions has broad applications in vegetation science.