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:In current work, we compared the separation of HeLa samples using two different trap columns for sample trapping, desalting, and preconcentration. The two columns have different types of the C18 stationary phase: a) a conventional packed PepMap C18 trap-column (Thermo Fischer Scientific), and b) the superficially porous silicon pillars μPAC C18 trap-column (PharmaFluidics)
Project description:The evolution of mass spectrometry-based proteomics has been driven by continuous advances in instrumentation, sample preparation, and data acquisition strategies. While chromatographic separation has historically been considered a critical bottleneck in achieving comprehensive proteome coverage, recent developments in ultra-fast mass spectrometry acquisition fundamentally challenge this paradigm. We investigated whether traditional chromatographic optimization principles established during the early era of proteomics remain essential in contemporary workflows. Using five distinct stationary phases, C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluated proteome identification performance using state-of-the-art data-independent acquisition on the Orbitrap Astral mass spectrometer with HeLa tryptic digests. Despite substantial chromatographic differences in selectivity and peak characteristics that would have profoundly influenced analytical outcomes in earlier instrumentation generations, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor and approximately 9,000 protein group identifications, regardless of column length variations. While distinct selectivity fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in bulk identification depth under high-speed acquisition conditions exceeding 200 Hz. From these findings we conclude that the analytical bottleneck has fundamentally shifted from chromatographic resolution to mass spectrometric sampling efficiency, where comprehensive peptide identification is now achieved through advanced spectral deconvolution rather than physical separation alone. This paradigmatic shift suggests that method development priorities in modern proteomics should evolve beyond traditional separation optimization to emphasize operational robustness, analytical throughput, and systematic reproducibility for routine applications.
Project description:The evolution of mass spectrometry-based proteomics has been driven by continuous advances in instrumentation, sample preparation, and data acquisition strategies. While chromatographic separation has historically been considered a critical bottleneck in achieving comprehensive proteome coverage, recent developments in ultra-fast mass spectrometry acquisition fundamentally challenge this paradigm. We investigated whether traditional chromatographic optimization principles established during the early era of proteomics remain essential in contemporary workflows. Using five distinct stationary phases, C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluated proteome identification performance using state-of-the-art data-independent acquisition on the Orbitrap Astral mass spectrometer with HeLa tryptic digests. Despite substantial chromatographic differences in selectivity and peak characteristics that would have profoundly influenced analytical outcomes in earlier instrumentation generations, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor and approximately 9,000 protein group identifications, regardless of column length variations. While distinct selectivity fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in bulk identification depth under high-speed acquisition conditions exceeding 200 Hz. From these findings we conclude that the analytical bottleneck has fundamentally shifted from chromatographic resolution to mass spectrometric sampling efficiency, where comprehensive peptide identification is now achieved through advanced spectral deconvolution rather than physical separation alone. This paradigmatic shift suggests that method development priorities in modern proteomics should evolve beyond traditional separation optimization to emphasize operational robustness, analytical throughput, and systematic reproducibility for routine applications.
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.