N-glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: a comparison

N-glycans of the mouse glycoprotein HSA and its human analogue CD24 from lymphoblastoma, neuroblastoma and astrocytoma cell lines as well as from mouse brain homogenate were analysed and compared to each other and to the N-glycosylation pattern of total glycoproteins from mouse and human brain. The N-glycans were released from PVDF-blotted HSA or CD24 and separated on Carbograph SPE into neutral and acid glycans. The naturally neutral glycan fraction and the fraction of glycans rendered neutral after neuraminidase treatment were analysed without further purification by MALDI-MS. In each fraction, about 25 molecular ions with an intensity >10% of the base peak were identified which corresponded to glycans with distinct isobaric monosaccharide compositions. Comparison of the neutral and desialylated glycans revealed some similarities between the samples analysed, but also clear differences. HSA and CD24 from all cell lines express almost no neutral N-glycans with two or more fucose in contrast to brain HSA and glycoproteins from mouse and human brain. The lack of extensive fucosylation was also observed for desialylated glycans of HSA and CD24 from all cell lines analysed except for CD24 from a human neuroblastoma cell line which exhibits like total human and mouse brain glycoproteins a large variety of highly fucosylated, higher branched N-glycans. HSA from mouse brain carries in addition desialylated non-fucosylated glycans of high abundance which were detected, if at all, only at low intensity in all other samples analysed suggesting that they may be implicated in specific functions of mouse brain HSA. Therefore, a rapid assessment of similarities or differences between glycosylation patterns of a glycoprotein isolated from different sources is possible using methods as described here.     

Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.     

Integrated view and comparative analysis of baseline protein expression in mouse and rat tissues

The increasingly large amount of proteomics data in the public domain enables, among other applications, the combined analyses of datasets to create comparative protein expression maps covering different organisms and different biological conditions. Here we have reanalysed public proteomics datasets from mouse and rat tissues (14 and 9 datasets, respectively), to assess baseline protein abundance. Overall, the aggregated dataset contained 23 individual datasets, including a total of 211 samples coming from 34 different tissues across 14 organs, comprising 9 mouse and 3 rat strains, respectively.In all cases, we studied the distribution of canonical proteins between the different organs. The number of canonical proteins per dataset ranged from 273 (tendon) and 9,715 (liver) in mouse, and from 101 (tendon) and 6,130 (kidney) in rat. Then, we studied how protein abundances compared across different datasets and organs for both species. As a key point we carried out a comparative analysis of protein expression between mouse, rat and human tissues. We observed a high level of correlation of protein expression among orthologs between all three species in brain, kidney, heart and liver samples, whereas the correlation of protein expression was generally slightly lower between organs within the same species. Protein expression results have been integrated into the resource Expression Atlas for widespread dissemination.     

Pilot study identifying myosin heavy chain 7, desmin,insulin‐like growth factor 7, and annexin A2 as circulating biomarkers of human heart failure

In‐depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac‐specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin‐like growth factor‐binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross‐reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150‐fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human.     

Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera

The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.     

Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain

Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.     

Reduced plasma APOA1 level is associated with Gastric Tumor Growth in MKN45 mouse xenograft model

There is no suitable diagnostic and prognostic biomarker for gastric cancer. The biggest hurdles in biomarker discovery are (i) the low abundance of cancer cell-specific proteins that limits their detection and (ii) complex inter-patient variations that complicate the discovery process. To circumvent these issues, we conducted proteomics on the plasma of gastric cancer mouse xenograft and attempted to identify proteins released by cancer cells. MKN45 gastric cancer cells were subcutaneously implanted into immune-incompetent nude mice. Plasma samples collected from mice with different tumor sizes (low, mid and high tumor loads) were subjected to iTRAQ and mass spectrometric analyses. Detection of human APOA1 in mouse plasma was verified and its expression level was shown to be lower in mice with large tumors compared to those with small tumors. Studies on a panel of about 14 gastric cancer cell lines supported the notion that APOA1 in mouse plasma was of human gastric cancer cell origin. While the clinical utility of APOA1 remains to be ascertained with a larger scale study, the current work supported the feasibility of using mouse xenograft model for gastric cancer biomarker discovery.     

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP)

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.     

Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human.     

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.     

Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics

Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias to some degree, assigned ranks among the techniques revealed that for most LC-FTICR-MS analyses linear regression normalization ranked either first or second. However, the lack of a definitive trend among the techniques suggested the need for additional investigation into adapting normalization approaches for label-free proteomics. Nevertheless, this study serves as an important step for evaluating approaches that address systematic biases related to relative quantification and label-free proteomics.     

Human and mouse cementum proteins immunologically related to enamel proteins

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.     

High-resolution analysis of salmonellae from turtles within a headwater spring ecosystem

Sediments and water from the pristine headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on the carapace and from the cloacae of 17 musk turtles (Sternotherus odoratus) and one snapping turtle (Chelydra serpentina serpentina) caught at the same site, were analysed for salmonellae by culture and molecular techniques. Whereas enrichment cultures from sediment and water samples were negative for salmonellae in PCR- and in situ hybridization-based analyses, both techniques detected salmonellae after enrichments from both carapace and cloacae of nine (i.e. of 53%) musk turtles. Further characterization of 10 isolates obtained from the enrichment cultures of four selected individuals and confirmed as salmonellae by PCR analysis was achieved by fingerprinting techniques (rep-PCR). The results show differences between individuals and, in one case, variation among isolates from a single individual. All isolates from two individuals displayed identical profiles. These profiles were different from those obtained from the isolates of the third individual, which were, themselves, also identical for all isolates. Salmonellae were much more diverse in samples from the carapace of the last individual with five different rep-PCR profiles retrieved. Serotyping of seven isolates representative for each rep-PCR profile identified all isolates as representing Salmonella enterica subspecies enterica serotype Rubislaw, which demonstrates the presence of different strains of potentially human pathogenic salmonellae naturally occurring on turtles even within pristine environments. The frequent detection of these organisms in biofilms on the carapace opens the door for speculations on the role of this habitat as a reservoir for salmonellae, and on potential implications for turtles acting as a dispersal vector.     

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson's disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.     

Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling

Loss of renal function is often associated with the injury of kidney glomeruli. It is therefore necessary to understand the mechanisms leading to progressive glomerular diseases; this may be addressed using proteomics. Until now, however, analysis of the glomeruli proteome using 2-DE has been technically hampered by low protein yields from scarce samples. To circumvent this problem, we developed a procedure which allows the human and mouse glomeruli proteome to be analysed. In this study, two different approaches were used to isolate mouse and human glomerular protein from kidney cortex. Mouse glomeruli were extracted by embolisation magnetic beads into the glomerular capillaries. Laser capture microdissection (LCM) was utilised to harvest glomeruli from human biopsy material. Human and murine samples were analysed using a fluorescence saturation labelling technique. Using 3 microg mouse glomerular protein a total of 2900 spots were resolved for differential proteome analysis. Moreover, it was also demonstrated for the first time that only ten glomeruli (0.5 microg) picked by LCM from a slide of a human kidney biopsy material were sufficient to visualise 900 spots. This novel strategy paves the way for future experiments aimed at investigating functional proteomics of glomerular diseases in humans and in mice.     

Assessing the use of thermal treatment to preserve the intact proteomes of post‐mortem heart and brain tissue

Protein degradation that occurs in tissue during post‐mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap‐freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2‐DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high‐molecular‐weight proteins and reduction in protein fragmentation.