Ancient proteins resolve the evolutionary history of Darwin's South American ungulates.

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

Spatially resolved analysis of small molecules by matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

• Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. • Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. • Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. • Finally, we discuss how MALDI-MSI can contribute to applied plant research.

MALDI-imaging mass spectrometry - An emerging technique in plant biology.

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research.

Proteome and flavonoid analysis reveals distinct responses of epidermal tissue and whole leaves upon UV-B radiation of barley (Hordeum vulgare L.) seedlings.

We describe here the effect of UV-B irradiation on the proteome and flavonoid content of the barley seedling leaf epidermis and mesophyll. Flavonoid analysis was performed using UPLC-PDA/-MS. The major flavonoid molecule responding to UV-B radiation was saponarin, and this accumulated in the epidermis, but not in the mesophyll. Changes in protein expression were determined using two-dimensional gel electrophoresis (2-DE) and identified 11 responsive proteins (seven up-regulated and two down-regulated in the epidermis; and one up- and one down-regulated in the mesophyll). A label-free LC-MS/MS(E) approach was applied for a subset of samples consisting of epidermis tissue and was able to detect a further 15 (11 up-regulated and four down-regulated) proteins. Most of the proteins with changed expression pattern after UV-B treatment were involved in initial responses characteristic for oxidative stress. Others were primary metabolism proteins involved in the supply of precursors for secondary metabolites. A separate analysis of epidermis and mesophyll tissue is important to give a spatially resolved picture of the response to UV-B treatment. The label-free LC-MS/MS(E) approach is complementary to the more conventional two-dimensional gel electrophoresis, as there was no overlap between the spectra of proteins identified by the two techniques.

Protein analysis of laser capture micro-dissected tissues revealed cell-type specific biological functions in developing barley grains.

Both the nucellar projection (NP) and endosperm transfer cells (ETC) of the developing barley grain (harvested 8 days after flowering) were isolated by laser capture micro-dissection combined with pressure catapulting. Protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF mass spectrometry. The majority of the ~160 proteins identified were involved in translation, protein synthesis, or protein destination. The NP proteome was enriched for stress defense molecules, while proteins involved in assimilate transport and the mobilization of nutrients were common to both the NP and the ETC. The combined qualitative and quantitative protein profiling allowed for the identification of several proteins showing tissue specificity in their expression, which underlines the distinct biological functions of these two tissues within the developing barley grain.

Protein and metabolite analysis reveals permanent induction of stress defense and cell regeneration processes in a tobacco cell suspension culture.

The secretome of a tobacco cell suspension culture was investigated by a combined proteomic and metabolomic approach. Protein analysis from 2-DE gels led to identification of 32 out of 60 spots from culture medium. Identified proteins were mainly involved in stress defence and cell regeneration processes. Among them three putative new isoforms, e.g. for chitinase, peroxidase and beta-1,4-xylosidase were identified, not yet present in available protein databases for the genus Nicotiana. GC-MS analysis of time course experiments revealed significant changes for metabolites involved in energy transport, signalling and cell development. Among them, the most significant increase was found for putrescine in the medium of cultures entering the exponential phase. Results showed strong abundance of stress associated proteins and metabolites in the absence of elicitors or additional stress treatments.

Salinity stress in roots of contrasting barley genotypes reveals time-distinct and genotype-specific patterns for defined proteins.

Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence, particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance on saline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are well characterized. A proteomic analysis of the roots of two contrasting cultivars (cv. Steptoe and cv. Morex) is presented. Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150 mM NaCl. The root proteome was analyzed based on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins were identified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysis grouped these into five clusters based on similarity of expression profile. The rank product method was applied to statistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinity tolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerous as-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis were detected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms and membrane stability via fine tuning of phytohormone and secondary metabolism in the root.

Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides.

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.