A Robust Protocol for Protein Extraction and Digestion.
Methods in molecular biology (Clifton, N.J.)
2017; 1550: 1-10
High-dimensional analysis of the aging immune system: Verification of age-associated differences in immune signaling responses in healthy donors
JOURNAL OF TRANSLATIONAL MEDICINE
Proteins play a key role in all aspects of cellular homeostasis. Proteomics, the large-scale study of proteins, provides in-depth data on protein properties, including abundances and post-translational modification states, and as such provides a rich avenue for the investigation of biological and disease processes. While proteomic tools such as mass spectrometry have enabled exquisitely sensitive sample analysis, sample preparation remains a critical unstandardized variable that can have a significant impact on downstream data readouts. Consistency in sample preparation and handling is therefore paramount in the collection and analysis of proteomic data.Here we describe methods for performing protein extraction from cell culture or tissues, digesting the isolated protein into peptides via in-solution enzymatic digest, and peptide cleanup with final preparations for analysis via liquid chromatography-mass spectrometry. These protocols have been optimized and standardized for maximum consistency and maintenance of sample integrity.
View details for DOI 10.1007/978-1-4939-6747-6_1
View details for PubMedID 28188518
Genomic relationship between SINE retrotransposons, Pol III- Pol II transcription, and chromatin organization: the journey from junk to jewel
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE
2011; 89 (5): 495-504
Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors.In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)].Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets.These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.
View details for DOI 10.1186/1479-5876-12-178
View details for Web of Science ID 000338475000001
View details for PubMedID 24952610
View details for PubMedCentralID PMC4229969
Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal
2011; 10 (17): 3016-3030
A typical eukaryotic genome harbors a rich variety of repetitive elements. The most abundant are retrotransposons, mobile retroelements that utilize reverse transcriptase and an RNA intermediate to relocate to a new location within the cellular genomes. A vast majority of the repetitive mammalian genome content has originated from the retrotransposition of SINE (100-300 bp short interspersed nuclear elements that are derived from the structural 7SL RNA or tRNA), LINE (7kb long interspersed nuclear element), and LTR (2-3 kb long terminal repeats) transposable element superfamilies. Broadly labeled as "evolutionary junkyard" or "fossils", this enigmatic "dark matter" of the genome possesses many yet to be discovered properties.
View details for DOI 10.1139/O11-046
View details for Web of Science ID 000299780400008
View details for PubMedID 21916613
View details for PubMedCentralID PMC3660135
Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called "master" pluripotency regulators.
View details for DOI 10.4161/cc.10.17.17543
View details for Web of Science ID 000294480700035
View details for PubMedID 21862875
View details for PubMedCentralID PMC3218602