Lisa Chung

Review of software tools for design and analysis of large scale MRM proteomic datasets

Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can be measured in a triple quadrupole instrument coupled to an LC, by a well-designed scheduling and selection of m/z windows. The design of a good MRM assay relies on the availability of peptide spectra from previous discovery-phase LC-MS/MS studies. The tedious aspect of manually developing and processing MRM assays involving thousands of transitions has spurred to development of software tools to automate this process. Software packages have been developed for project management, assay development, assay validation, data export, peak integration, quality assessment, and biostatistical analysis. No single tool provides a complete end-to-end solution, thus this article reviews the current state and discusses future directions of these software tools in order to enable researchers to combine these tools for a comprehensive targeted proteomics workflow.

Early-Life Stress Perturbs Key Cellular Programs in the Developing Mouse Hippocampus

Conflicting reports are available with regard to the effects of childhood abuse and neglect on hippocampal function in children. While earlier imaging studies and some animal work have suggested that the effects of early-life stress (ELS) manifest only in adulthood, more recent studies have documented impaired hippocampal function in maltreated children and adolescents. Additional work using animal modes is needed to clarify the effects of ELS on hippocampal development. In this regard, genomic, proteomic, and molecular tools uniquely available in the mouse make it a particularly attractive model system to study this issue. However, very little work has been done so far to characterize the effects of ELS on hippocampal development in the mouse. To address this issue, we examined the effects of brief daily separation (BDS), a mouse model of ELS that impairs hippocampal-dependent memory in adulthood, on hippocampal development in 28-day-old juvenile mice. This age was chosen because it corresponds to the developmental period in which human imaging studies have revealed abnormal hippocampal development in maltreated children. Exposure to BDS caused a significant decrease in the total protein content of synaptosomes harvested from the hippocampus of 28-day-old male and female mice, suggesting that BDS impairs normal synaptic development in the juvenile hippocampus. Using a novel liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) assay, we found decreased expression of many synaptic proteins, as well as proteins involved in axonal growth, myelination, and mitochondrial activity. Golgi staining in 28-day-old BDS mice showed an increase in the number of immature and abnormally shaped spines and a decrease in the number of mature spines in CA1 neurons, consistent with defects in synaptic maturation and synaptic pruning at this age. In 14-day-old pups, BDS deceased the expression of proteins involved in axonal growth and myelination, but did not affect the total protein content of synaptosomes harvested from the hippocampus, or protein levels of other synaptic markers. These results add two important findings to previous work in the field. First, our findings demonstrate that in 28-day-old juvenile mice, BDS impairs synaptic maturation and reduces the expression of proteins that are necessary for axonal growth, myelination, and mitochondrial function. Second, the results suggest a sequential model in which BDS impairs normal axonal growth and myelination before it disrupts synaptic maturation in the juvenile hippocampus.

Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation

Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for addiction treatment.

Development of a Targeted Urine Proteome Assay for kidney diseases

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases.

Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins

We present a comprehensive workflow for large scale (>1000 transitions/run) label‐free LC‐MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC‐MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label‐free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC‐MRM assay.

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

Use of a Targeted Urine Proteome Assay (TUPA) to identify protein biomarkers of delayed recovery after kidney transplant

Development of delayed graft function (DGF) following kidney transplant is associated with poor outcomes. An ability to rapidly identify patients with DGF versus those with immediate graft function (IGF) may facilitate the treatment of DGF and the research needed to improve prognosis. The purpose of this study was to use a Targeted Urine Proteome Assay to identify protein biomarkers of delayed recovery from kidney transplant.