Biology subjects

Pandi, B.

Publications and source records attributed to Pandi, B..

6 recordsLinked to original sources

bioRxiv2026

Tracking cellular biomolecular condensate dynamics under proteostatic stress with middle-down phosphoproteomics

Coordination of biological function requires the partition of cellular components including into biomolecular condensates, but an overall landscape of how protein compartmentalize into higher-order assemblies under stress is still emerging. We apply proteome-wide solubility profiling to compare the compositions of NP-40-insoluble proteins and their phosphorylation status, using a new mass spectrometry-based hybrid bottom-up and chemical middle-down proteomics approach to analyze the solubility behavior of 8,740 proteins and 31,647 phosphopeptides under normal and ER stress conditions. Cell stress induces a pervasive differential partition of proteins in and out of detergent-insoluble cellular compartments. This differential partition is partially orthogonal to stress-induced abundance changes and comprises both phosphorylation-dependent and phosphorylation-independent mechanisms. Whereas phosphorylation-independent partition changes involve largely secretory pathway proteins and implicate higher-order assemblies of chaperones and clients, phosphorylation-based partitions suggest a dynamic rearrangement of biomolecular condensate compositions across cytoplasmic and nuclear ribonucleoprotein assemblies. The accumulation of serine/arginine rich (SR) proteins and other annotated nuclear speckle members in the condensate-rich proteome fractions emerges as a central feature of stress-induced remodeling. Our results establish global solubility dynamics as an integral component of proteome stress response and implicates broad involvements of splice factor spatial reorganization as a prominent facet of ER stress response.

biochemistry↗

bioRxiv2026

Proteogenomic analysis of the differential stability of cardiac protein isoforms

Alternative splicing is an important regulatory layer in gene expression, but knowledge on the isoform protein molecules continue to lag their canonical counterparts. An open question is whether alternative protein isoforms feature different half-life than the canonical counterpart, which could indicate differential usage and functional diversification. Here we combined a proteogenomics approach with heavy water-based protein turnover analysis to survey 24 pairs of canonical-alternative protein isoforms in the mouse heart. The results provide a reference on their numerical half-life and also reveal widespread differences in isoform stability.

biochemistry↗

bioRxiv2025

Micro- and macro- heterogeneity of N-glycoproteome remodeling in endoplasmic reticulum stress

N-glycosylation plays essential roles in the folding, trafficking, and maturation of proteins in the secretory pathways, but how individual protein and residue glycosylation rewires under endoplasmic reticulum (ER) stress is unknown. Particularly, intact glycopeptide data that retain the connectivity between glycosylation sites and the attached glycans are needed to reveal the micro- and macro- heterogeneity of N-glycosylation sites and their permutations in stressed cells. Here, we developed an optimized magnetic polyethyleneimine boronic acid-containing scaffold (mPBA) enrichment workflow to achieve sensitive and broad enrichment of intact glycoproteins for mass spectrometry analysis, quantifying 13759 unique protein-, site-, and glycoform combinations, termed glycopeptidoforms, in normal and stressed cells while requiring only 0.1 to 0.5 mg total peptide input. The data reveals a systems-level shift in the fate of hundreds of glycoproteins. N-glycosylation changes are highly dynamic, with magnitude far exceeding protein expression changes, and showing complex protein-, site-, and glycan-specific granularity. Individual glycoform reconfigurations can be observed that suggest lesions within specific steps in protein maturation and trafficking pathways. Mannose trimming is disrupted across multiple proteins and cell states, suggesting a central feature of ER stress mediated glycoproteome remodeling. Together, these results reveal molecular details into the remodeling of protein secretory pathways upon ER stress and highlight the utility of mPBA for sensitive N-glycoproteomics studies.

biochemistry↗

bioRxiv2025

Deuterium labeling enables proteome wide turnover kinetics analysis in cell culture

The half-life of proteins is tightly regulated and underlies many cellular processes. It remains unclear the extent to which proteins are dynamically synthesized and degraded in different cell types and cell states. We introduce an improved D2O labeling workflow and apply it to examine the landscape of protein turnover in pluripotent and differentiating human induced pluripotent stem cells (hiPSC). The majority of hiPSC proteins show minimal turnover beyond cell doubling rates, but we also identify over 100 new fast-turnover proteins not previously described as short-lived. These include proteins that function in cell division and cell cycle checkpoints, that are enriched in APC/C and SPOP degrons, and that are depleted upon pluripotency exit. Differentiation rapidly shifts the set of fast-turnover proteins toward including RNA binding and splicing proteins. The ability to identify fast-turnover proteins in different cell cultures also facilitates secretome analysis, as exemplified by studies of hiPSC-derived cardiac myocytes and primary human cardiac fibroblasts. The presented workflow is broadly applicable to protein turnover studies in diverse primary, pluripotent, and transformed cells.

biochemistry↗

bioRxiv2024

Improved determination of protein turnover rate with heavy water labeling by mass isotopomer ratio selection

The synthesis and degradation rates of proteins form an essential component of gene expression control. Heavy water labeling has been used in conjunction with mass spectrometry to measure protein turnover rates, but the optimal analytical approaches to derive turnover rates from the isotopomer patterns of deuterium labeled peptides continue to be a subject of research. Here we describe a method, which comprises a reverse lookup of numerically approximated peptide isotope envelopes, coupled to the selection of optimal isotopomer pairs based on peptide sequence, to calculate the molar fraction of new peptide synthesis in heavy water labeling mass spectrometry experiments. We validated this approach using an experimental calibration curve comprising mixtures of fully unlabeled and fully labeled proteomes. We then re-analyzed 17 proteome-wide turnover experiments from four mouse organs, and showed that the method increases the coverage of well-fitted peptides in protein turnover experiments by 25-82%. The method is implemented in the Riana software tool for protein turnover analysis, and may avail ongoing efforts to study the synthesis and degradation kinetics of proteins in animals on a proteome-wide scale. Whats newWe describe a reverse lookup method to calculate the molar fraction of new synthesis from numerically approximated peptide isotopomer profiles in heavy water labeling mass spectrometry experiments. Using an experimental calibration curve comprising mixtures of fully unlabeled and fully labeled proteomes at various proportions, we show that this method provides a straightforward way to calculate the proportion of new proteins in a protein pool from arbitrarily chosen isotopomer ratios. We next analyzed which of the isotopomer pairs within the peptide isotope envelope yielded isotopomer time courses that fit most closely to kinetic models, and found that the identity of the isotopomer pair depends partially on the number of deuterium accessible labeling sites of the peptide. We next derived a strategy to automatically select the isotopomer pairs to calculate turnover rates based on peptide sequence, and showed that this increases the coverage of existing proteome-wide turnover experiments in multiple data sets of the mouse heart, liver, kidney, and skeletal muscle by up to 25-82%.

biochemistry↗

bioRxiv2023

Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart

A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H). Examined variant regions that differ between alternative and canonical isoforms are highly enriched in intrinsically disordered regions, and over two-thirds of such regions are predicted to function in protein binding and/or RNA binding. The analysis here lends further credence to the notion that alternative splicing diversifies the proteome by rewiring intrinsically disordered regions, which are increasingly recognized to play important roles in the generation of biological function from protein sequences.

biochemistry↗