GNPS - 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:Chromatographic column selection can impact proteomic profiling, yet comparative studies remain limited. Here, we evaluate the performance of a conventional flame-pulled Accucore resin-packed capillary column and a microfabricated pillar array column (μPAC) in a sample multiplexed global proteome profiling experiment using six human cell lines prepared in triplicate as a TMTpro18-plex. Overall, the chromatography columns exhibited comparable performance. Specifically, the number and overlap of quantified peptides, as well as proteins, was similar between columns. Principal component and hierarchical clustering analyses highlighted reproducible patterns of cell line organization, while correlation analyses showed high replicate consistency across column formats. Similarity, analytical parameters like XCorr scores, signal-to-noise ratio, and peak resolution showed consistency. These findings demonstrate the potential for using robust, standardized microfluidic columns, such as μPAC, in lieu of traditional pull-tipped capillary columns without sacrificing depth or quantitative accuracy. Key advantages of μPAC include its ease of use and durability in a uniform format, although this advantage does come at a higher cost. This comparative analysis offers valuable insights into column selection for TMT-based quantitative proteomics.
Project description:This SuperSeries is composed of the following subset Series: GSE37664: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 1A) GSE37670: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 2A) GSE37826: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 2C) Refer to individual Series
Project description:The effective separation of complex peptide mixtures is a cornerstone of mass spectrometry-based proteomics analysis that enhances the accuracy and depth of proteomic analyses. Here we compare datasets collected of whole cell tryptic peptides which were fractionated by either conventional flame-pulled, C18 packed bed microcapillary columns or microfabricated pillar array columns (μPAC). Sixteen samples from four yeast strains (Δmet6, Δpfk2, Δura2, and wildtype) were analyzed in quadruplicate using data-independent acquisition (DIA). Each column enabled the quantification of >4,700 protein, with >95.4% overlap between column formats. The μPAC showed higher MS1 and MS2 signal intensities, while maintaining similar peptide characteristics as the capillary column. The capillary column favored slightly longer and more hydrophobic peptides. Both columns achieved high data completeness at the protein level (>95%) and reproducible quantification, with μPAC offering slight improvements. Principal component analysis and correlation analysis confirmed the capture of yeast strain-specific differences, with hierarchical clustering prioritizing strain over column effects. Protein quantification validated gene knockouts in both column formats, demonstrating similar accuracy of quantification. These findings highlight the μPAC as a standardized and robust alternative to capillary columns in proteomic analysis.
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.