Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

Metabolomics identifies pyrimidine starvation as the mechanism of 5-aminoimidazole-4-carboxamide-1-ß-riboside-induced apoptosis in multiple myeloma cells.

To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl-α-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce multiple myeloma cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in multiple myeloma cells.

Mass spectral measurements of the apoHDL in horse (Equus caballus) cerebrospinal fluid.

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468Da that we are speculating is the value for horse apoE.

Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL.

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.

Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase ß1 subunits.

N-Glycans of the Na,K-ATPase ß1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the ß1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous ß1 subunit with various YFP-linked ß1 subunits expressed in Madin-Darby canine kidney cells was used to assess ß1-ß1 interactions. The amount of co-precipitated endogenous dog ß1 was greater with dog YFP-ß1 than with rat YFP-ß1, showing that amino acid-mediated interactions are important for ß1-ß1 binding. Co-precipitation of ß1 was also less with the unglycosylated YFP-ß1 than with glycosylated YFP-ß1, indicating a role for N-glycans. Mixing cells expressing dog YFP-ß1 with non-transfected cells increased the amount of co-precipitated ß1, confirming the presence of intercellular (YFP-ß1)-ß1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated ß1 subunits. The decrease in ß1 co-precipitation both with rat YFP-ß1 and unglycosylated YFP-ß1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-ß1)-ß1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the ß1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the ß1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.

PYY(1-36) is the major form of PYY in rat distal small intestine: quantification using high-resolution mass spectrometry.

We measured molecular forms of PYY in the distal half of rat small intestine using a new method for tissue extraction, three sequential reverse phase chromatography steps, and PYY radioimmunoassay and mass spectrometry to measure their levels. The extraction method called RAPID, developed to minimize artifactual degradation of PYY during tissue extraction and sample preparation, uses Reduced temperature, Acidified buffer, Peptidase inhibitors, Isotopically enriched mass spectrometry standards, and Dilution to inhibit and monitor endogenous peptide degradation during tissue processing. Synthetic peptides [PYY(1-36)-NH(2), PYY(3-36)-NH(2), PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH] selectively enriched with (13)C(3)-alanine were added as internal standards to the extraction buffer. By collecting mass spectra rather than multiple-reaction-monitoring (MRM) profiles, we simultaneously screen for any PYY forms that were present in the immunoreactive fractions. PYY(1-36)-NH(2), PYY(3-36)-NH(2), PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH were identified and quantified at 64.3±4.5, 6.1±0.9, 0.9±0.1, and <0.3pmol/g of tissue, respectively (n=3). Thus, we found that in rat distal small intestine proPYY is processed to PYY(1-36)-NH(2) with little conversion to PYY(3-36)-NH(2). These data suggest that production of PYY(3-36)-NH(2) (a form with greater potency than PYY(1-36)-NH(2) for inhibition of feeding and gastric emptying) occurs after the peptide leaves its cell of synthesis by enzymatic action in the circulation.

Detection of two distinct forms of apoC-I in great apes.

ApoC-I, the smallest of the soluble apolipoproteins, associates with both TG-rich lipoproteins and HDL. Mass spectral analyses of human apoC-I previously had demonstrated that in the circulation there are two forms, either a 57 amino acid protein or a 55 amino acid protein, due to the loss of two amino acids from the N-terminus. In our analyses of the apolipoproteins of the other great apes by mass spectrometry, four forms of apoC-I were detected. Two of these showed a high degree of identity to the mature and truncated forms of human apoC-I. The other two were homologous to the virtual protein and its truncated form that are encoded by a human pseudogene. In humans, the genes for apoC-I and its pseudogene are located on chromosome 19, the pseudogene being 2.5 kb downstream from the apoC-I gene. Based on the similarity between the apoC-I gene and the pseudogene, it has been concluded that the latter arose from the former as a result of gene duplication approximately 35 million years ago. Interestingly, the virtual protein encoded by the pseudogene is acidic, not basic like apoC-I. In the chimpanzee, there also are two genes for apoC-I, the one upstream encodes a basic protein and the downstream gene, rather than being a pseudogene, encodes an acidic protein (P86336). In addition to reporting on the molecular masses of great ape apoC-I, we were able to clearly demonstrate by "Top-down" sequencing that the acidic form arose from a separate gene. In our analyses, we have measured the molecular masses of apoC-I associated with the HDL of the following great apes: bonobo (Pan paniscus), chimpanzee (Pan troglodytes), and the Sumatran orangutan (Pongo abelii). Genomic variations in chromosome 19 among great apes, baboons and macaques as they relate to both genes for apoC-I and the pseudogene are compared and discussed.

Micro-heterogeneity of human saliva Peptide P-C characterized by high-resolution top-down Fourier-transform mass spectrometry.

Top-down proteomics characterizes protein primary structures with unprejudiced descriptions of expressed and processed gene products. Gene sequence polymorphisms, protein post-translational modifications, and gene sequence errors can all be identified using top-down proteomics. Saliva offers advantages for proteomic research because of availability and the noninvasiveness of collection and, for these reasons, is being used to search for disease biomarkers. The description of natural protein variants, and intra- and inter-individual polymorphisms, is necessary for a complete description of any proteome, and essential for the discovery of disease biomarkers. Here, we report a striking example of natural protein variants with the discovery by top-down proteomics of two new variants of Peptide P-C. Intact mass measurements, and collisionally activated-, infrared multiphoton-, and electron capture-dissociation, were used for characterization of the form predicted from the gene sequence with an average mass 4371 Da, a form postulated to result from a single nucleotide polymorphism of mass 4372 Da, and another form of mass 4370 Da postulated to arise from a novel protein sequence polymorphism. While the biological significance of such subtle variations in protein structure remains unclear, their importance cannot be assigned without their characterization, as is reported here for one of the major salivary proteins.

Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation.

Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to today's bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14-261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (-17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N(6)-(retinylidene) modification (266.20 Da) between residues 225-248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b(6)f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane beta-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane alpha-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.

Rpe65 isomerase associates with membranes through an electrostatic interaction with acidic phospholipid headgroups.

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

Hepatic lipase maturation: a partial proteome of interacting factors.

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.

Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins.

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human­chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.

A new endogenous form of PYY isolated from canine ileum: Gly-extended PYY(1-36).

We purified and identified the peptide YY (PYY) forms present and determined their levels from a portion of the canine ileum directly adjacent to the cecum by a new extraction method designed to prevent and evaluate degradation of endogenous peptides. We used three reverse phase chromatography steps with radioimmunoassay of fractions for PYY-like-immunoreactivity (PYY-LI). The purified fractions underwent intact protein/peptide mass spectrometry identification and sequencing (i.e. "top-down" MS analysis). This analysis confirmed the identity of a new form of PYY, PYY(1-36)-Gly, which co-elutes with PYY(1-36)-NH(2) through all three of separation steps used. The PYY(1-36)-Gly form represents approximately 20% of the total PYY found in this region of the canine intestine. In addition, we also found that the PYY(3-36)-NH(2) form represents 6% of the total PYY in the canine ileo-cecal junction. The physiological implication of the Gly-extended form of PYY(1-36) warrants further investigation.

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Mass spectral analysis of the apolipoproteins on dog (Canis lupus familiaris) high density lipoproteins. Detection of apolipoprotein A-II.

In a recent study, we reported the detection of apoA-II associated with the plasma high density lipoproteins of pigs that were previously thought to lack or to have this apolipoprotein in trace amounts. Dogs have also been reported to lack this apolipoprotein; however, genomic data have revealed that the gene for apoA-II is present on chromosome 38. Prompted by this finding, we have carried out detailed mass spectral measurements on dog apo HDL. The molecular mass of apoA-II was obtained as well as values for proapoA-I, apoA-I, apoC-I. In each case, the measured values were found to be in excellent agreement with the corresponding molecular weights calculated from genomic data. Following reverse-phase chromatography, tryptic fragments in selected fractions were analyzed by tandem mass spectrometry (MSMS). In addition to apoA-I, proapoA-I and apoA-II, enzymatic fragments from both apoC-II and apoA-IV were detected. Post-translational modification (PTM) of apoA-I, involving glycosylation, oxidation of a single methionine and acylation, were also noted. We also report on the sequencing of apoC-I using "Top Down" mass spectrometry analysis.

Characterization of the outer membrane proteome of Leptospira interrogans expressed during acute lethal infection.

Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infection in rats. Recent studies have demonstrated that protein and lipopolysaccharide constituents of Leptospira recovered from acutely infected guinea pig tissue differ from that of Leptospira in chronically infected rat tissue and in vitro cultivated Leptospira (J. E. Nally, E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett, Infect. Immun. 73:3251-3260, 2005). In the current study, the proteome of Leptospira expressed during disease processes was characterized relative to that of in vitro cultivated Leptospira (IVCL) after enrichment for hydrophobic membrane proteins with Triton X-114. Protein samples were separated by two-dimensional gel electrophoresis, and antigens expressed during infection were identified by immunoblotting with monospecific antiserum and convalescent rat serum in addition to mass spectrometry. Results suggest a significant increase in the expression of the outer membrane protein Loa22 during acute infection of guinea pigs relative to other outer membrane proteins, whose expression is generally diminished relative to expression in IVCL. Significant amounts of LipL32 are also expressed by Leptospira during acute infection of guinea pigs.

Protein-Sequence Polymorphisms and Post-translational Modifications in Proteins from Human Saliva using Top-Down Fourier-transform Ion Cyclotron Resonance Mass Spectrometry.

Single nucleotide polymorphisms (SNPs) can result in protein sequence polymorphisms (PSPs) when codon translations are altered. Both top-down and bottom-up proteomics strategies can identify PSPs, but only if databases and software are used with this in mind. A 14319 Da protein from human saliva was characterized using the top-down approach on a hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer equipped for both collisionally-activated (CAD) and electron-capture (ECD) dissociation. Sequence tags identified the protein as Cystatin SN, and defined the N-terminal signal peptide cleavage site, as well as two disulfide bonds, in agreement with previous studies. The mass of the intact protein (< 5 ppm error) deviated from that calculated from the published gene sequence by 16.031 Da, and, based on CAD and ECD fragment ion assignments, it was concluded that the isoform of the protein analyzed carried a PSP at residue 11 such that the Pro translated from the genome was in fact Leu/Ile. An independently determined SNP (rs2070856) subsequently confirmed the genetic basis of the mass spectral interpretation and defined the residue as Leu. In another example, the PRP3 protein with mass ∼10,999 Da was found to be an isomeric/isobaric mixture of the reported sequence with PSPs D4N or D50N (rs1049112). Both CAD and ECD datasets support two phosphorylation sites at residues Ser8 and Ser22, rather than Ser17. In the context of discovery proteomics, previously undefined PSPs and PTMs will only be detected if the logic of data processing strategies considers their presence in an unbiased fashion.

Pentose phosphate cycle oxidative and nonoxidative balance: A new vulnerable target for overcoming drug resistance in cancer.

The metabolic network of cancer cells confers adaptive mechanisms against many chemotherapeutic agents, but also presents critical constraints that make the cells vulnerable to perturbation of the network due to drug therapy. To identify these fragilities, combination therapies based on targeting the nucleic acid synthesis metabolic network at multiple points were tested. Results showed that cancer cells overcome single hit strategies through different metabolic network adaptations, demonstrating the robustness of cancer cell metabolism. Analysis of these adaptations also identified the maintenance of pentose phosphate cycle oxidative and nonoxidative balance to be critical for cancer cell survival and vulnerable to chemotherapeutic intervention. The vulnerability of cancer cells to the imbalance on pentose phosphate cycle was demonstrated by phenotypic phase plane analysis.

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications.

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.