Project description:This is the third of three MassIVE repositories associated with the manuscript "Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day." This repository contains 2,130 single PBMC measurements (71 raw files) acquired using a dual-column LC system with real-time library searching (RTLS). [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs from a healthy donor were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Protein concentration was determined by BCA, followed by dilution and sequential digestion with Lys-C and trypsin (1:50 enzyme-to-protein ratios). Peptides were desalted using C18 MacroSpin columns, concentrated, quantified, and dried prior to labeling. [TMTpro Labeling of Bulk Bridge Sample]: Bulk PBMC peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 and 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL for use as a bridge sample. [Single-Cell Isolation (cellenONE)]: PBMCs were thawed, washed, and resuspended in PBS before single-cell isolation using a cellenONE instrument. Cells were selected based on size, circularity, and elongation to exclude debris, apoptotic cells, and doublets. Individual cells were dispensed into 30 wells of 32-plex N2 nanoPOTS chips, with two wells reserved for bridge sample addition. Chips were stored at -80 deg C until processing. [N2 nanoPOTS Single-Cell Sample Preparation]: Single cells were digested overnight at 37 deg C in nanoliter volumes using TMT-compatible buffer containing Lys-C and trypsin. TMTpro 32-plex reagents were dispensed directly into each well using the cellenONE. Labeling proceeded overnight, followed by quenching with hydroxylamine, acidification with formic acid, evaporation, and storage at -20 deg C. TMTpro-labeled bridge sample was added at levels described in the manuscript. [LC-MS/MS Analysis with RTLS]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer with FAIMS. Dual parallel columns enabled near-continuous MS operation. Peptides were separated at 100 nL/min using a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were applied. RTLS was implemented using rapid ion-trap MS2 scans searched in real time against FAIMS-specific spectral libraries. Precursors passing a cosine similarity threshold >= 40 triggered high-resolution Orbitrap MS2 acquisition. Orbitrap MS1 scans were acquired at 120,000 resolution, followed by HCD fragmentation and Orbitrap MS2 detection for RTLS-selected precursors. [Database Searching and Quantification]: All raw files were processed using FragPipe v23.0 with MSFragger, MSBooster, Percolator, Philosopher, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24). Only high-resolution Orbitrap scans were retained for database searching. Search parameters included 20 ppm precursor tolerance, semi-tryptic specificity, static TMTpro modification on lysine and peptide N-termini, and methionine oxidation as a variable modification. Protein-level FDR was set to 1%. TMTpro quantification was performed using IonQuant and TMT-Integrator with median aggregation and MS1-based ratio-to-abundance conversion.
Project description:This experiment describes the quantitative proteomic profiling of whole-cell, soma, and neuronal projection fractions from day 35 induced neurons (iN) derived from control and ASAH1 knockout human embryonic stem cells. Soma and projection compartments were physically separated using Transwell culture inserts, and triplicate biological replicates were processed using TMTpro 18-plex labeling and high-resolution Orbitrap mass spectrometry with FAIMS. Data were searched against the human proteome with stringent FDR control and reporter ion quantification, enabling compartment-resolved analysis of proteome alterations associated with ASAH1 deficiency.
Project description:Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ~30% gain in protein identification for short gradients (120 SPD and 80 SPD). However, even the lowest tested tissue amount (0.04 nL, 40,000 µm3) yielded 3,200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7,500 proteins from 0.5 nL of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research.
Project description:Longitudinal samples were collected from neonates in the NICU at the Royal Women’s Hospital in Melbourne, Australia. Blood collection occurred by heel stick and was collected on Whatman paper shortly after birth at 25 weeks gestation, one day post birth, and at the equivalent of 28, 32, 36, and 40 weeks gestation.
Project description:This experiment describes a quantitative proteomic analysis of whole-cell lysates from induced neurons (iN) and induced dopaminergic neurons (iDA) differentiated from human embryonic stem cells of three genotypes: control, ASAH1 knockout, and SMPD1 knockout. Triplicate biological samples were prepared and analyzed using TMTpro 18-plex labeling coupled to high-resolution Orbitrap mass spectrometry with FAIMS. Data were processed with target-decoy database searching, stringent FDR control, and reporter ion–based quantification. The resulting dataset enables comparative profiling of proteomic alterations associated with lysosomal dysfunction and neurodegeneration-relevant pathways in distinct neuronal subtypes.
Project description:The kidney differentiation protocol takes induced pluripotent stem cells through to kidney organoid via directed differentiation in approximately 25 days. The cells are grown in a monolayer in a dish for seven days and are subjected to growth factors before being pelleted on day seven. The organoids then continue to differentiate as a 3D structure, with at least 8 distinct kidney cell types identifiable around day 18. This data has been generated to investigate the reproducibility of the kidney differentiation protocol using RNA sequencing from day 18 organoids. Organoid differentiations were performed in two separate batches separated in time. The resulting RNA-seq data was analysed to identify genes with highly variable expression between batches.
Project description:CosMx SMI is a high-plex in situ analysis platform to provide spatial multiomics with formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tissue samples at cellular and subcellular resolution. CosMx SMI is an integrated system with mature cyclic fluorescent in situ hybridization (FISH) chemistry, high-resolution imaging readout, interactive data analysis and visualization software. Herein we used the CosMx SMI with the CosMxTM Human Universal Cell Characterization RNA Panel (1000-plex) supplemented with 14 custom genes of interest to probe different disease stages of MASLD/MASH, previously known as NAFLD.
Project description:The field of proteomics has evolved hand-in-hand with technological advances in LC-MS/MS systems, now enabling the analysis of very deep proteomes in a reasonable time. However, most applications do not deal with full cell or tissue proteomes, but rather with restricted sub-proteomes relevant for the research context at hand or resulting from extensive fractionation. At the same time, investigation of many conditions or perturbations puts a strain on measurement capacity. Here we develop a high throughput workflow capable of dealing with large numbers of low or medium complexity samples and specifically aim at the analysis of 96-well plates in a single day (15 min per sample). We combine parallel sample processing with a modified liquid chromatography platform driving two analytical columns in tandem, which are coupled to a quadrupole Orbitrap mass spectrometer (Q Exactive HF). The modified LC platform eliminates idle-time between measurements and the very high sequencing speed of the Q Exactive HF dramatically reduces required measurement time. We apply the pipeline to the yeast chromatin remodeling landscape, and demonstrate quantification of 96 pull-downs of chromatin complexes in about one day. This is achieved with only 500 µg input material, enabling yeast cultivation in a 96-well format. Our system retrieved known complex-members and the high throughput allowed probing with many bait proteins. Even alternative complex compositions were detectable in these very short gradients. Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved several-fold compared to established workflows. The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.
Project description:Comparison of murine colonic mucosal gene expression between postanatal day 90 (P90) to postnatal day 30 (P30) by whole genomic expression microarray. Gene expression profiling of colonic mucosal DNA between P90 and P30 mice. Agilent Technologies two-color labelling kit and genomic hybridization protocol was utilized.
Project description:Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid isotope labeling strate-gies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quanti-tative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample losses, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotop-ic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of bio-logical and clinical proteomics applications.