Martha Stapels

Proteorhodopsin in the ubiquitous marine bacterium SAR11

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 (‘Pelagibacter ubique’), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents.

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co‐immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5‐signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule‐associated protein 2a and dynamin 1, as mGluR5‐interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.

Polycomb Group Proteins as Epigenetic Mediators of Neuroprotection in Ischemic Tolerance

Polycomb group proteins protect neurons from injury through a mechanism involving decreased potassium channel function. Promoting Tolerance A temporary or permanent interruption of blood flow (ischemia) to the brain can lead to brain injury, with potentially devastating consequences, such as stroke. Intriguingly, previous exposure to a brief period of ischemia that is not itself sufficient to cause brain injury can protect against a later, more prolonged period of ischemia. This phenomenon, called ischemic tolerance, is associated with a general suppression of gene expression in the tolerant brain. Paradoxically, the tolerant state depends on protein synthesis. Stapels et al. performed proteomic analyses of mouse brains subjected to various ischemic conditions and found, in ischemic-tolerant brains, an increase in the abundance of polycomb group proteins, which act as transcriptional repressors. Further analyses of both mouse brain and cultured cells indicated that the polycomb proteins SCMH1 and BMI1 inhibited potassium channel abundance and activity and that this represented a mechanism underlying ischemic tolerance. A further understanding of the mechanisms underlying tolerance could open the door to new therapies for ischemic stroke. Exposing the brain to sublethal ischemia affects the response to a subsequent, otherwise injurious ischemia, resulting in transcriptional suppression and neuroprotection, a response called ischemic tolerance. Here, we show that the proteomic signature of the ischemic-tolerant brain is characterized by increased abundance of transcriptional repressors, particularly polycomb group (PcG) proteins. Knocking down PcG proteins precluded the induction of ischemic tolerance, whereas in an in vitro model, overexpressing the PcG proteins SCMH1 or BMI1 induced tolerance to ischemia without preconditioning. We found that PcG proteins are associated with the promoter regions of genes encoding two potassium channel proteins that show decreased abundance in ischemic-tolerant brains. Furthermore, PcG proteins decreased potassium currents in cultured neuronal cells, and knocking down potassium channels elicited tolerance without preconditioning. These findings reveal a previously unknown mechanism of neuroprotection that involves gene repressors of the PcG family.

Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome

Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42–75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MSE-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT1A receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.

Comprehensive characterization of the N-glycosylation status of CD44s by use of multiple mass spectrometry-based techniques

The CD44 family are type-1 transmembrane glycoproteins which are important in mediating the response of cells to their microenvironment, including regulation of growth, survival, differentiation, and motility. All these important functions have been reported to be regulated by N-glycosylation; however, little is known about this process. In the CD44 family, the most prolific isoform is CD44 standard type (CD44s). In this work, an integrated strategy combining stable isotope labeling, chemical derivatization, hydrophilic-interaction liquid chromatographic (HILIC) separation, and mass spectrometric (MS) identification was used to perform a comprehensive qualitative and quantitative survey of the N-glycosylation of recombinant CD44s. Specifically, the occupation ratios of the N-glycosites were first determined by MS with 18O labeling; the results revealed five glycosites with different occupation ratios. Next, N-glycans were profiled by chemical derivatization and exoglycosidase digestion, followed by MALDI–TOF-MS and HILIC–ESI–MS–MS analysis. Interestingly, the quantitative analysis showed that non-sialylated, fucosylated complex-type glycans dominated the N-glycans of CD44s. Furthermore, the site-specific N-glycan distributions profiled by LC–ESI–MSE indicated that most glycosites bore complex-type glycans, except for glycosite N100, which was occupied by high-mannose-type N-glycans. This is the first comprehensive report of the N-glycosylation of CD44s.FigureStrategies for characterization of the N-glycosylation status of CD44s