Peter Berndt

Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints.

Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry of protein samples from two‐dimensional (2‐D) gels in conjunction with protein sequence database searches is frequently used to identify proteins. Moreover, the automatic analysis of complete 2‐D gels with hundreds and even thousands of protein spots (“proteome analysis”) is possible, without human intervention, with the availability of highly accurate mass spectrometry instruments, and high‐throughput facilities for preparation and handling of protein samples from 2‐D gels. However, the lack of software for precise automatic analysis and annotation of mass spectra, as well as software for in‐batch sequence database queries, is increasingly becoming a significant bottleneck for the proteomics work flow. In the present paper we outline an algorithm for reliable, accurate, and automatic evaluation of mass spectrometric data and database searches. We show here that simply selecting from the sequence database the protein that has the most matching fragment masses often leads to false‐positive results. Reliable protein identification is dependent on several parameters: the accuracy of fragment mass determination, the number of masses submitted for query, the mass distribution of query masses, the number of masses matching between sample and database protein, the size of the sequence database, and the kind and number of modifications considered. Using these parameters, we derive a simple statistical estimation that can be used to calculate the probability of true‐positive protein identification.

Two-dimensional map of human brain proteins.

Samples of human brain from the parietal cortex lobe were analyzed by two‐dimensional gel electrophoresis, using immobilized pH gradient strips covering the various pH regions. The protein spots were visualized with colloidal Coomassie blue stain and identified by matrix‐assisted laser desorption/ionization mass spectrometry. Approximately 400 spots were identified, corresponding to 180 different brain proteins. The list of identified proteins includes a large number of structural proteins and of enzymes or enzyme subunits with various catalytic activities. The majority of proteins are localized in the cytoplasma and in mitochondria. The two‐dimensional map may be useful as a reference database to study changes in the protein level caused by various disorders, such as Alzheimers disease, major depression and schizophrenia.

Identification of Nicotinamide N-Methyltransferase as a Novel Serum Tumor Marker for Colorectal Cancer

Purpose: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm. Experimental Design: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples. Results: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-β induced protein ig-h3 (βIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78. Conclusions: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.

Two-dimensional map of the proteome of Haemophilus influenzae

We have constructed a two‐dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two‐dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low‐copy‐number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope‐bound protein fraction using either immobilized pH gradient strips or a two‐detergent system with a cationic detergent in the first and an anionic detergent in the second‐dimensional separation. Different proteins (502) were identified by matrix‐assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two‐dimensional proteome databases.

Identification of PSME3 as a Novel Serum Tumor Marker for Colorectal Cancer by Combining Two-dimensional Polyacrylamide Gel Electrophoresis with a Strictly Mass Spectrometry-based Approach for Data Analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550–6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

Metabolite Profiling of Alzheimer's Disease Cerebrospinal Fluid

Alzheimers disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine.

Two-dimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3-acetamidophenol

Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP‐induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two‐dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP‐induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3‐acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two‐dimensional (2‐D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10—50% of the control value and were more marked in the high‐ than in the low‐dose of APAP‐treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N‐acetyl‐p‐benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP‐induced hepatotoxicity.

Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.

Visualization of low‐copy‐number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low‐abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two‐dimensional electrophoresis and identified by matrix‐assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two‐dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low‐abundance but also major components of the E. coli protein extract. In particular, it enriched many low‐molecular‐mass proteins, such as cold‐shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat‐ and cold‐shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two‐dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.

Depletion of the high-abundance plasma proteins

Summary.Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.

The rat liver mitochondrial proteins.

Subcellular fractionation increases the probability of detection of low‐abundance proteins. We prepared a fraction highly enriched in mitochondrial proteins from rat liver. The proteins were analyzed by two‐dimensional (2‐D) electrophoresis using broad‐and narrow‐range immobilized pH gradient strips, and identified by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). 192 different gene products were detected, of which approximately 70% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Eight gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 10–15 spots corresponded to one gene product.