Faster, reduced cost calibration method development methods for the analysis of fermentation product using near-infrared spectroscopy (NIRS).

Recent innovations in synthetic biology, fermentation, and process development have decreased time to market by reducing strain construction cycle time and effort. Faster analytical methods are required to keep pace with these innovations, but current methods of measuring fermentation titers often involve manual intervention and are slow, time-consuming, and difficult to scale. Spectroscopic methods like near-infrared (NIR) spectroscopy address this shortcoming; however, NIR methods require calibration model development that is often costly and time-consuming. Here, we introduce two approaches that speed up calibration model development. First, generalized calibration modeling (GCM) or sibling modeling, which reduces calibration modeling time and cost by up to 50% by reducing the number of samples required. Instead of constructing analyte-specific models, GCM combines a reduced number of spectra from several individual analytes to produce a large pool of spectra for a generalized model predicting all analyte levels. Second, randomized multicomponent multivariate modeling (RMMM) reduces modeling time by mixing multiple analytes into one sample matrix and then taking the spectral measurements. Afterward, individual calibration methods are developed for the various components in the mixture. Time saved from the use of RMMM is proportional to the number of components or analytes in the mixture. When combined, the two methods effectively reduce the associated cost and time for calibration model development by a factor of 10.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae.

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae.

We observed that removing pantothenate (vitamin B5), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce ß-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, ß-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a "metabolic switch" for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.

Probing conformational changes in Ape1 during the progression of base excision repair.

Abasic (AP) sites are the most common lesions arising in genomic DNA. Repair of this potentially mutagenic DNA damage is initiated by the major apurinic/apyrimidinic endonuclease Ape1, which specifically recognizes and cleaves the DNA backbone 5' to the AP site. Ape1 is one of the major proteins in the base excision repair pathway (BER), and deletions in any of the BER proteins result in embryonic lethality. In this study, we employed fluorescence spectroscopy and in vitro mass spectrometric protein footprinting to investigate Ape1 conformational changes during various nucleoprotein interactions along its reaction pathway. Differences in intrinsic fluorescence emission spectra were observed during Ape1 protein's processing of the substrate, indicating possible conformational changes of the nucleoprotein complexes. To determine the protein domains that are involved in the putative conformational change, full-length Ape1 protein was probed with a lysine-reactive reagent (NHS-biotin) in the context of free protein and DNA-bound complexes. Protection patterns between pre- and postincision complexes revealed an increased susceptibility of lysine residues localized on the Ape1 surface that contacts the 3' end of the incised duplex (downstream of the incision site). We propose that the decreased protection results from Ape1 having a more relaxed grip on this section of the incised duplex to facilitate the handoff to the downstream BER enzyme. Protection of lysines (residues 24-35) in the N-terminal region was also observed in the intact AP-DNA-bound complex. These residues are part of the Ref1 domain which functions to regulate the activity of several transcription factors but to date has not been ascribed a DNA binding function. The reactivity of these Ref1 lysines was restored in the postincision complex. The differential protection patterns of lysines in the flexible N-terminal domain suggest a novel Ref1 conformational change concomitant with DNA binding and catalysis. It is likely that Ape1 employs this structural switch to mediate redox and nuclease activities. The ability of the Ape1-AP-DNA complex to recruit other BER proteins was also investigated by probing ternary complexes comprised of Ape1, DNA polymerase beta (Polbeta), and different BER DNA intermediates (abasic or gapped DNA). Our results suggest that Polbeta approaches the Ape1-DNA complex downstream of the incision site, displaces Ape1 DNA binding contacts (K227, K228, and K276), and in the process makes minimal interactions with lysine residues in the Ref1 domain.

Post-translational modifications of Desulfovibrio vulgaris Hildenborough sulfate reduction pathway proteins.

Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications (PTMs). Although PTMs are a critical aspect of cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit (DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium, Desulfovibrio desulfuricans G20, also showed similar +42 Da modifications in the same pathway. Here, we discuss our methods and implications of potential trimethylation in the D. vulgaris sulfate reduction pathway.

Use of a designed peptide array to infer dissociation trends for nontryptic peptides in quadrupole ion trap and quadrupole time-of-flight mass spectrometry.

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.

Influence of crosslinker identity and position on gas-phase dissociation of Lys-Lys crosslinked peptides.

A systematic study of the dissociation patterns of crosslinked peptides analyzed by tandem mass spectrometry is reported. A series of 11-mer peptides was designed around either a polyalanine or polyglycine scaffold with arginine at the C terminus. One or two lysine residues were included at various locations within the peptides to effect inter- or intra-molecular crosslinking, respectively. Crosslinked species were generated with four commonly used amine-specific chemical crosslinking reagents: disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), dithiobis(succinimidylpropionate) (DSP), and disuccinimidyl glutarate (DSG). The influence of precursor charge state, location of crosslink, and specific crosslinking reagent on the MS/MS dissociation pattern was examined. Observed trends in the dissociation patterns obtained for these species will allow for improvements to software used in the automated interpretation of crosslinked peptide MS/MS data.

Expanded coverage of the human heart mitochondrial proteome using multidimensional liquid chromatography coupled with tandem mass spectrometry.

Recent evidence suggests that mitochondria are closely linked with the aging process and degenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, there has been increasing interest in cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We have previously reported results of a one-dimensional electrophoresis/liquid chromatography MS/MS study to characterize the proteome of normal human heart mitochondria (Taylor et al. Nat. Biotechnol. 2003, 21, 281-286). We now report two subsequent studies where multidimensional liquid chromatography MS/MS was investigated as an alternative means for characterizing the same sample.

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells.

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.

Characterization of the human heart mitochondrial proteome.

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

Conformational studies of Zn-ligand-hexose diastereomers using ion mobility measurements and density functional theory calculations.

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0-90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal-ligand-monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.

An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria.

An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.