Biology subjects

Post, M. A.

Publications and source records attributed to Post, M. A..

3 recordsLinked to original sources

bioRxiv2025

Automation of high-throughput arrayed lentivirus production and titration

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 106 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x105 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

microbiology↗

bioRxiv2023

Maximizing glycoproteomics results through an integrated PASEF workflow

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics is evolving rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of Parallel Accumulation Serial Fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using the timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width and the use of do-pant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 minutes, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics condition to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/570555v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c89a72org.highwire.dtl.DTLVardef@113dc50org.highwire.dtl.DTLVardef@e52daborg.highwire.dtl.DTLVardef@1951b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

N-linked glycosylation of the M-protein variable region: Glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma.

Multiple Myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.

cancer biology↗