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Dive into the research topics where Gregg Czerwieniec is active.

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Featured researches published by Gregg Czerwieniec.


Analytical Chemistry | 2012

Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions.

Chang Ho Sohn; Heather D. Agnew; J. Eugene Lee; Michael J. Sweredoski; Robert L. Graham; Geoffrey T. Smith; Sonja Hess; Gregg Czerwieniec; Joseph A. Loo; James R. Heath; Raymond J. Deshaies; J. L. Beauchamp

We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.


Journal of the American Chemical Society | 2012

Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification

Chang Ho Sohn; J. Eugene Lee; Michael J. Sweredoski; Robert L. Graham; Geoffrey T. Smith; Sonja Hess; Gregg Czerwieniec; Joseph A. Loo; Raymond J. Deshaies; J. L. Beauchamp

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.


Glia | 2016

Traumatically injured astrocytes release a proteomic signature modulated by STAT3-dependent cell survival.

Jaclynn Levine; Eunice Kwon; Pablo M. Paez; Weihong Yan; Gregg Czerwieniec; Joseph A. Loo; Michael V. Sofroniew; Ina-Beate Wanner

Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch‐injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure‐pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e., mechanoporation and cell lysis. This acute trauma‐release proteome was overrepresented with metabolic proteins compared with the uninjured cellular proteome, bearing relevance for post‐traumatic metabolic depression. Astrocyte‐specific deletion of signal transducer and activator of transcription 3 (STAT3‐CKO) resulted in reduced stretch‐injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3‐CKO versus nontransgenic astrocytes. STAT3‐CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC), and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3‐dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and were robustly expressed in reactive astrocytes 1 day postinjury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among postinjury astrocytes. These findings reveal a proteomic signature of traumatically‐injured astrocytes reflecting STAT3‐dependent cellular survival with potential diagnostic value. GLIA 2016;64:668–694


Journal of Cerebral Blood Flow and Metabolism | 2017

New astroglial injury-defined biomarkers for neurotrauma assessment

Julia Halford; Sean Shen; Kyohei Itamura; Jaclynn Levine; Albert C. Chong; Gregg Czerwieniec; Thomas C. Glenn; David A. Hovda; Paul Vespa; Ross Bullock; W. Dalton Dietrich; Stefania Mondello; Joseph A. Loo; Ina B. Wanner

Traumatic brain injury (TBI) is an expanding public health epidemic with pathophysiology that is difficult to diagnose and thus treat. TBI biomarkers should assess patients across severities and reveal pathophysiology, but currently, their kinetics and specificity are unclear. No single ideal TBI biomarker exists. We identified new candidates from a TBI CSF proteome by selecting trauma-released, astrocyte-enriched proteins including aldolase C (ALDOC), its 38kD breakdown product (BDP), brain lipid binding protein (BLBP), astrocytic phosphoprotein (PEA15), glutamine synthetase (GS) and new 18-25kD-GFAP-BDPs. Their levels increased over four orders of magnitude in severe TBI CSF. First post-injury week, ALDOC levels were markedly high and stable. Short-lived BLBP and PEA15 related to injury progression. ALDOC, BLBP and PEA15 appeared hyper-acutely and were similarly robust in severe and mild TBI blood; 25kD-GFAP-BDP appeared overnight after TBI and was rarely present after mild TBI. Using a human culture trauma model, we investigated biomarker kinetics. Wounded (mechanoporated) astrocytes released ALDOC, BLBP and PEA15 acutely. Delayed cell death corresponded with GFAP release and proteolysis into small GFAP-BDPs. Associating biomarkers with cellular injury stages produced astroglial injury-defined (AID) biomarkers that facilitate TBI assessment, as neurological deficits are rooted not only in death of CNS cells, but also in their functional compromise.


Analytical Chemistry | 2004

Reagentless Detection and Classification of Individual Bioaerosol Particles in Seconds

David P. Fergenson; Maurice Pitesky; Herbert J. Tobias; Paul T. Steele; Gregg Czerwieniec; Scott C. Russell; Carlito B. Lebrilla; Joanne Horn; Keith R. Coffee; Abneesh Srivastava; Segaran P. Pillai; Meng Ta Peter Shih; Howard L. Hall; Albert J. Ramponi; John T. Chang; Richard G. Langlois; Pedro L. Estacio; Robert T. Hadley; Matthias Frank; Eric E. Gard


Analytical Chemistry | 2003

Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry.

Paul T. Steele; Herbert J. Tobias; David P. Fergenson; Maurice Pitesky; Joanne Horn; Gregg Czerwieniec; Scott C. Russell; Carlito B. Lebrilla; and Eric E. Gard; Matthias Frank


Analytical Chemistry | 2005

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry.

Abneesh Srivastava; Maurice Pitesky; Paul T. Steele; Herbert J. Tobias; David P. Fergenson; Joanne Horn; Scott C. Russell; Gregg Czerwieniec; Carlito B. Lebrilla; Eric E. Gard; Matthias Frank


Analytical Chemistry | 2005

Achieving high detection sensitivity (14 zmol) of biomolecular ions in bioaerosol mass spectrometry

Scott C. Russell; Gregg Czerwieniec; Carlito B. Lebrilla; Paul T. Steele; Vincent J. Riot; Keith R. Coffee; Matthias Frank; Eric E. Gard


Analytical Chemistry | 2005

Stable Isotope Labeling of Entire Bacillus atrophaeus Spores and Vegetative Cells Using Bioaerosol Mass Spectrometry

Gregg Czerwieniec; Scott C. Russell; Herbert J. Tobias; Maurice Pitesky; David P. Fergenson; Paul T. Steele; Abneesh Srivastava; Joanne Horn; Matthias Frank; Eric E. Gard; Carlito B. Lebrilla


Journal of the American Society for Mass Spectrometry | 2006

Structural relationships in small molecule interactions governing gas-phase enantioselectivity and zwitterionic formation

Xin Cong; Gregg Czerwieniec; Erica L. McJimpsey; Seonghee Ahn; Frederic A. Troy; Carlito B. Lebrilla

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Matthias Frank

Lawrence Livermore National Laboratory

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Paul T. Steele

Lawrence Livermore National Laboratory

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Eric E. Gard

Lawrence Livermore National Laboratory

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David P. Fergenson

Lawrence Livermore National Laboratory

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Joanne Horn

Lawrence Livermore National Laboratory

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Abneesh Srivastava

Lawrence Livermore National Laboratory

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