HIV-Associated Neurocognitive Disorder Column Test
Ontology highlight
ABSTRACT: Plasma and Cerebrospinal fluid data were acquired on the Q-Exactive with 3 different columns: a C8 column, a C18 column, and a polar C18 Column.
Project description:Plasma and Cerebrospinal fluid data were acquired on the Q-Exactive with 3 different columns: a C8 column, a C18 column, and a polar C18 Column.
Project description:Although the benefits of reduction of the size of reversed phase particles are established to provide increased sequencing depth and improved chromatography in LCMS experiments, the wide-scale adoption of optimally sized small particles in reversed-phase columns has been hampered by the necessity for specialized equipment such as ultra-high pressure liquid chromatography or a customized column heating apparatus. Here, we introduce a new strategy to routinely fabricate a 50 cm-long, 1.9 µm particle C18 column and extensively characterize the performance of this column. This column was packed under 100 Bar and routinely utilized on a standard quarternary HPLC at pressures below 300 Bar. Expanding the depth of sequencing of peptides that show a statistically significant quantitative change arising from a biological stimulation is critical. Compared with traditional C18 columns packed with 3 µm particles, the column with the 1.9 µm particles operated with a standard HPLC could detect 330% more peptides with statistically significant changes from differentially stimulated T cells. This improved column fabrication methodology provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize sequencing depth, dynamic range, sensitivity, and reproducibility. This study also highlights the importance of the statistical analysis of quantitative proteomic data instead of a sole focus on peptide spectrum match yields.
Project description:Fe-IMAC columns for robust and reproducible phosphopeptide ernichment, comparison to TiO2 batch and Ti-IMAC tip enrichment, large scale phosphoproteomics coupling Fe-IMAC column pre-enrichment to subsequent hSAX separation
Project description:A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yield-ed impressive results, current studies suffer from a limited proteomic depth and dynamic range there-fore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Our data shows that µPAC columns clearly outperform classical packed bed columns boosting peptide IDs by up to 50% and protein IDs by up to 24%. Using the above-mentioned analysis platform, more than 10,000 proteins could be identified from a single 2 h gradient shotgun analysis for a triple proteome mix of human, yeast and E. coli digests. At high sample loads of 400 ng all three uPAC types yielded comparable number of protein identifications, whereas the 50cm neo column performed best when lower inputs of less than 200 ng were injected. This additional dataset comprises additional data generated with the Aurora Elite G3 column (150 mm x 75 µm, 1.7 µm, IonOpticks) for comparison to the aforementioned µPAC technology.
Project description:Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies aim to systematically catalogue, characterize, and comprehend alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics workflows. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but it remained a bottleneck for methods that require higher throughput or a scale that is beyond the maximum capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference in both the number and properties of the phosphopeptides identified when using either method. In all, we illustrate how scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA for global phosphoproteome profiling.