Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.     

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.     

Localization of peroxisomal matrix proteins by photobleaching

The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.     

Identification of new Golgi complex specific proteins by direct organelle proteomic analysis

The Golgi complex is in the crossroad of the endocytic and secretory pathways. Its function is to post-translationally modify and sort proteins and lipids, and regulate the membrane balance in the cell. To understand the structure-function relationship of the Golgi complex the Golgi proteome has to be identified first. We have used a direct organelle proteomic analysis to identify new Golgi complex proteins. Enriched stacked Golgi membrane fractions from rat livers were isolated, and the proteins from these membranes were subsequently digested into peptides. The peptides were fractionated by cation-exchange chromatography followed by protein identification by automated capillary-LC/ESI-MS/MS analysis and database searches. Two different search programs, ProID and MASCOT were used. This resulted in a total of 1125 protein identifications in two experiments. In addition to the known Golgi resident proteins, a significant number of unknown proteins were identified. Some of these were further characterized in silico using different programs to provide insight into their structure, intracellular localization and biological functions. The Golgi localization of two of these newly identified proteins was also confirmed by indirect immunofluorescence.     

The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast)

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes. Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established. Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome. It was identified to be plastid ribosome recycling factor. Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.     

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast)

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.     

Mass tagging approach for mitochondrial thiol proteins

A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.     

Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.     

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.     

Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.     

Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

We developed a strategy to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation-microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.     

Multiple epitope tagging of expressed proteins for enhanced detection

Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7. The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system. The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells. In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

Technical improvement to 2D-PAGE of rice organelle membrane proteins

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.