The (15)N isotope effect as a means for correlating phenotypic alterations and affected pathways in a trait anxiety mouse model.

Stable isotope labeling techniques hold great potential for accurate quantitative proteomics comparisons by MS. To investigate the effect of stable isotopes in vivo, we metabolically labeled high anxiety-related behavior (HAB) mice with the heavy nitrogen isotope (15)N. (15)N-labeled HAB mice exhibited behavioral alterations compared to unlabeled ((14)N) HAB mice in their depression-like phenotype. To correlate behavioral alterations with changes on the molecular level, we explored the (15)N isotope effect on the brain proteome by comparing protein expression levels between (15)N-labeled and (14)N HAB mouse brains using quantitative MS. By implementing two complementary in silico pathway analysis approaches, we were able to identify altered networks in (15)N-labeled HAB mice, including major metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Here, we discuss the affected pathways with regard to their relevance for the behavioral phenotype and critically assess the utility of exploiting the (15)N isotope effect for correlating phenotypic and molecular alterations.

Profiling of mouse synaptosome proteome and phosphoproteome by IEF.

Synapses play important roles in neurotransmission and neuroplasticity. For an in-depth analysis of the synaptic proteome and phosphoproteome, synaptosomal proteins from whole mouse brain were analyzed by IEF and MS resulting in the largest synaptosome proteome described to date, with 2980 unique proteins identified with two or more peptides. At the same time, 118 synaptosomal phosphoproteins were identified, eight of which are reported for the first time as phosphorylated. Expression of selected proteins in synaptosomes was investigated by Western blot. We demonstrate that IEF is a powerful method to interrogate complex samples such as brain tissue both at the proteome and the phosphoproteome level without the need of additional enrichment for phosphoproteins. The detailed synaptoproteome data set reported here will help to elucidate the molecular complexity of the synapse and contribute to our understanding of synaptic systems biology in health and disease.

Life-style changes of a halophilic archaeon analyzed by quantitative proteomics.

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.

Quantitative peptide and protein profiling by mass spectrometry.

Proteomics may be defined as the systematic analysis of proteins expressed in a given organism (Electrophoresis 16:1090-1094, 1995). Important technical innovations in mass spectrometry (MS), protein identification methods, and database annotation, over the past decade, now make it possible to routinely identify thousands of proteins in complex biological samples (Nature 422:198-207, 2003). However, to gain new insights regarding fundamental biological questions, accurate protein quantification is also required. In this chapter, we present methods for the biochemical separation of different cellular compartments, two-dimensional chromatographic separation of the constituent peptide populations, and the recently published Spectral Counting Strategy, a label-free MS-based protein quantification technology (Cell 125:173-186, 2006; Anal Chem 76:4193-4201, 2004; Mol Cell Proteomics 4:1487-1502, 2005; Cell 125:1003-1013, 2006; Methods 40:135-142, 2006; Anal Chem 77:6218-6224, 2005; J Proteome Res 5:2339-2347, 2006). Additionally, highly accurate protein quantification based on isotope dilution, describing the isotope coded protein label (ICPL) -- method shall be explained in detail (Mol Cell Proteomics 5:1543-1558, 2006; Proteomics 5:4-15, 2005).

Identification of the arginine/ornithine antiporter ArcD from Halobacterium salinarum.

This paper identifies the first arginine/ornithine antiporter ArcD from the domain of archea. The functional role of ArcD is demonstrated by transport assays with radioactive labelled arginine, by its necessity to enable arginine fermentation under anaerobic growth conditions and by the consumption of arginine from the medium during growth. All three experimentally observables are severely disturbed when the deletion strain DeltaArcD is used. The isolated protein is verified by mass spectrometry and reconstituted in vesicles. The proteoliposomes are attached to a membrane and capacitive currents are recorded which appear upon initiation of the transport process by change from arginine-free to arginine-containing buffer. This clearly demonstrates that the purified 34kD protein is the functional unit.

Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis.

Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and that-in contrast to current knowledge-Nalpha-acetylation is common in the archaeal domain of life with 13-18% of the proteins being Nalpha-acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.

The low molecular weight proteome of Halobacterium salinarum.

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.

Genome-wide proteomics of Natronomonas pharaonis.

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. According to genome analysis, the theoretical proteome consists of 2843 proteins. To reach further conclusions about its cellular physiology, the cytosolic protein inventory of Nmn. pharaonis has been analyzed using MS/MS on an ESI-Q-TOF mass spectrometer coupled on-line with a nanoLC system. The efficiency of this shotgun approach is illustrated by the identification of 929 proteins of which 886 are soluble proteins representing 41% of the cytosolic proteome. Cell lysis under denaturing conditions in water with subsequent separation by SDS-PAGE prior to nanoLC-MS/MS resulted in identification of 700 proteins. The same number (but a different subset) of proteins was identified upon cell lysis under native conditions followed by size fractionation (retaining protein complexes) prior to SDS-PAGE. Additional size fractionation reduced sample complexity and increased identification reliability. The set of identified proteins covers about 60% of the cytosolic proteins involved in metabolism and genetic information processing. Many of the identified proteins illustrate the high genetic variability among the halophilic archaea.

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey.

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.

Quantitative profiling of the membrane proteome in a halophilic archaeon.

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.

The membrane proteome of Halobacterium salinarum.

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation.

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).