Cornelia Koy

Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue

As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Molecular adaptations of Helicoverpa armigera midgut tissue under pyrethroid insecticide stress characterized by differential proteome analysis and enzyme activity assays.

Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry‐based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI‐TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS‐PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C‐III0, a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C‐III0 as potential key‐marker of the here proposed IUGR proteome signature, as it is a very low‐density lipoprotein (VLDL) and high‐density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Mass spectrometric proteome analysis suggests anaerobic shift in metabolism of Dauer larvae of Caenorhabditis elegans.

The Dauer larva is a non-feeding alternative larval stage of some nematodes specialized for long-term survival and dispersal. In this study we compared proteome maps obtained from Dauer larvae with those from the corresponding third larval stage (L3) of the feeding life cycle of C. elegans wild-type strain N2. We demonstrate at the protein level that altered metabolism may participate in longevity determination of Dauers. We detected huge amounts of alcohol dehydrogenase (CE12212) and aldehyde dehydrogenase (CE29809) in Dauer animals, indicating highly active fermentative pathways. Inorganic pyrophosphatase (CE05448) that enables to metabolize pyrophosphate as a high-energy source was over-expressed in Dauers. An interesting differentially expressed protein was phosphatidylethanolamine-binding protein (CE38516) that was found in high abundance in samples from Dauer larvae. Protein synthesis may be lowered in Dauer animals by the reduced expression of splicing factor rsp-3 (CE31089) and methionyl-tRNA synthase (CE34219). We observed significantly lower amounts of the pepsin-like aspartyl protease 1 (CE21681) in non-feeding Dauers, which is in agreement with reduced nutrient digestion. Finally, the hypothetical protein R08E5.2 (CE33294) was present in high abundance in L3 animals.

Mass spectrometric and peptide chip epitope mapping of rheumatoid arthritis autoantigen RA33.

The protein termed RA33 was determined to be one major autoantigen in rheumatoid arthritis (RA) patients and antiRA33 auto antibodies were found to appear shortly after onset of RA. They are often detectable before a final diagnosis can be made in the clinic. The aim of our study is to characterise the epitope of a monoclonal antiRA33 antibody on recombinant RA33 using mass spectrometric epitope mapping. Recombinant RA33 has been subjected to BrCN cleavage and fragments were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequent in-gel proteolytic digestion and mass spectrometric analysis determined the partial sequences in the protein bands. Western blotting of SDS-PAGE-separated protein fragments revealed immuno-positive, i.e. epitope-containing bands. BrCN-derived RA33 fragments were also separated by high-performance liquid chromatography (HPLC) and immuno-reactivity of peptides was measured by dot–blot analysis with the individual HPLC fractions after partial amino acid sequences were determined. The epitope region identified herewith was compared to data from peptide chip analysis with 15-meric synthetic peptides attached to a glass surface. Results from all three analyses consistently showed that the epitope of the monoclonal antiRA33 antibody is located in the aa79–84 region on recombinant RA33; the epitope sequence is MAARPHSIDGRVVEP. Sequence comparisons of the 15 best scoring peptides from the peptide chip analysis revealed that the epitope can be separated into two adjacent binding parts. The N-terminal binding parts comprise the amino acid residues “DGR”, resembling the general physico–chemical properties “acidic/polar–small–basic”. The C-terminal binding parts contain the amino acid residues “VVE”, with the motif “hydrophobic–gap–acidic”. The matching epitope region that emerged from our analysis on both the full-length protein and the 15-meric surface bound peptides suggests that peptide chips are indeed suitable tools for screening patterns of autoantibodies in patients suffering from autoimmune diseases.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples

AbstractPatient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients’ antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope–antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen–antibody interactions in order to improve disease diagnostic, we suggest that “protein-wide” screening for specific epitope–paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen–antibody interactions remain distinguishable. Figure 

Mass spectrometric profiling of cord blood serum proteomes to distinguish infants with intrauterine growth restriction from those who are small for gestational age and from control individuals.

Intrauterine growth restriction (IUGR) is a multifactorial condition in that the fetus does not reach its genetically given growth potential. Besides its contribution to perinatal mortality, it is a risk factor for cardiovascular and metabolic diseases later in life. The diagnosis is based on antenatal sonography, which allows differentiating between IUGR and fetuses that are small by constitution (small for gestational age [SGA]). Yet, neither a clinical nor a biochemical tool is available to confirm reliably the diagnosis of IUGR postnatally. Recently, we identified umbilical cord blood proteins of the apolipoprotein family by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with differential signal intensities between the IUGR group and a control group. We hypothesized that identified molecules have the potential to generate a proteome profile specific for IUGR. A total of 114 serum samples (42 from the IUGR group, 12 from the SGA group, and 60 from the control group) of the umbilical vein (99 samples) and umbilical artery (15 samples) were analyzed. Sample quality was estimated by determining the abundance of hemoglobin (hemolysis) and CXC-motif chemokines CXCL4 and CXCL7 (platelet activation). Samples passing the quality criteria were forwarded to multiplex apolipoprotein profiling. Assay performance was tested with the sample sets, resulting in a sensitivity of 0.91 and a specificity of 0.85 in the test set with venous blood samples. Arterial cord blood samples followed the trend (sensitivity, 0.67; specificity, 0.85). SGA samples grouped together with the control samples. We conclude that the proteome profiling signature is confirmatory to clinical surveillance with the potential to identify neonates with IUGR postnatally in low-birth weight babies born at uncertain gestational age when antenatal sonography data have not been recorded.

MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy – a pilot study

Identification of proteins from apheresis samples was performed by both SDS‐PAGE and 2‐D gel separation of eluted proteins from staphylococcal protein A‐based immunoadsorption columns (Prosorba®) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate‐spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2‐D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba® columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non‐redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL‐associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full‐length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A‐based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba® therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin‐related reactions during immunoadsorption therapy.