Project description:Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.
Project description:We recently demonstrated that perfluorooctanoic acid (PFOA), a volatile surfactant, is as effective as sodium dodecyl sulfate at solubilizing the membrane proteins. PFOA can be removed by repeated evaporation prior to mass spectrometry analysis. However, the removal of PFOA by evaporation is a lengthy process that takes approximately 6 h. Toward the goal of decreasing the length of time required to remove PFOA from protein digests, we tested the efficiency of PFOA removal and subsequent peptide recovery using strong cation exchange (SCX) chromatography, hydrophilic interaction chromatography (HILIC), fluorous solid phase extraction (FSPE), and anion exchange (ANX) chromatography. We found that all these chromatographic techniques except ANX chromatography remove PFOA thoroughly from protein digest. Peptide recovery rates from the SCX chromatography varied widely; nonacidic peptides were recovered at a rate of up to 95%, while acidic peptides were recovered at a rate of less than 10%. On the other hand, acidic peptides were recovered well from HILIC, while peptides whose pIs are greater than 6 were recovered poorly. Peptide recovery using FSPE was considerably lower, less than 10% for most of the peptides. These results indicate that the SCX and HILIC chromatography provide a more rapid alternative to the evaporation method for applications in which recovery of entire set of peptides is not required.
Project description:Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.
Project description:NMR-based metabolomics is a powerful tool to comprehensively monitor chemical processes in biological systems. Key to its success is the accurate and complete metabolite identification and quantification. Due to the inherent complexity of most metabolic mixtures, NMR peak overlap can make data analysis of 1D or even 2D NMR spectra challenging, especially for the 1H spectral region from 3.2-4.5 ppm that is dominated by carbohydrates and their derivatives. To address this problem, we present an effective method for carbohydrate signal removal in complex metabolomics samples by oxidation via the addition of sodium periodate (NaIO4). In an optional step, reaction products can be removed with hydrazide beads. The treated samples show substantially simplified 1D and 2D NMR spectra with their carbohydrate peaks removed, whereas noncarbohydrate peaks remain mostly unaffected. This allows the unrestricted detection of those metabolites that are otherwise obscured by carbohydrate signals. The method was first tested for metabolite model mixtures and then applied to urine and serum samples. It revealed a significant number of noncarbohydrates that were made unambiguously observable and identifiable by this method. The proposed protocol is simple and it is suitable for high-throughput sample treatment for the comprehensive metabolite identification in a broad range of samples.
Project description:BackgroundClinical diagnoses of fungal infections often rely upon culture techniques followed by microscopic examination of positive cultures and histopathological specimens. Culturing of microorganisms is prone to false negatives, while microscopy methods can be complicated by atypical phenotypes and organisms that are morphologically indistinguishable in tissues. Delays in diagnoses (or the lack thereof) and inaccurate identification of infectious organisms contribute to increased morbidity and mortality in patients.MethodsTwo-hundred randomized, heterogeneous patient blood and respiratory samples that were culture-negative were tested using polymerase chain reaction (PCR) amplification of internal transcribed spacer regions of ribosomal RNA genes utilizing panfungal primers. Amplicons were sequenced, subjected to sequence similarity searches, and compared using phylogenetic analyses.ResultsThirteen fungal sequences were detected in three whole-blood samples and nine respiratory samples. Bioinformatic analyses were performed which indicated the presence of multiple pathogens and potential pathogens.ConclusionsThe results from this pilot study demonstrate the utility of PCR assays and sequence analyses in clinical tests for fungi to facilitate rapid diagnosis and appropriate treatments to deal with the false negatives from culture results.
Project description:Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. Here we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient loss of dendritic spines. Spines destined for removal show local calcium accumulations and are subsequently removed by invading macrophages or activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.
Project description:Cell membrane proteins are densely decorated with surface glycosylation and intracellular phosphorylation whose interplay determines the cell-cell communication and signaling cascades. However, their concomitant characterization remains extreme challenges due to complexity of glycan structures, low abundance of glycopeptides (particularly sialylation), labile, dynamic nature and low detectability of both phosphopeptides and glycopeptides in mass spectrometry. In addition, the interplay and dynamic change between glycosylation and phosphorylation in tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells is still unclear. In this study, we introduce a streamlined metal ion-decorated ZIC-cHILIC strategy, featuring a simple pH control, to allow simultaneous enrichment and stepwise separation of intact (sialo-)glycopeptides and phosphopeptides.
Project description:Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer's recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of "representative samples" is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis.