18 research outputs found
Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions
We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and proteinâprotein 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
Dilution of oral D3âCreatine to measure creatine pool size and estimate skeletal muscle mass: development of a correction algorithm
Abstract Background Muscle mass can be measured directly in vivo by isotope dilution, using Creatineâ(methylâd3) monohydrate (D3âCr) by mouth followed by measurement of the steadyâstate enrichment of D3âcreatinine (D3âCrn) in urine. Isotope dilution methods require knowledge of the amount of tracer delivered to the pool of interest. In a subset of human subjects, a small amount of orally administered D3âCr âspillsâ into urine after absorption and prior to transport into skeletal muscle cells. The objectives were to develop a method to correct for spillage to compare the estimate of muscle mass by D3âCr dilution to other assessments of fatâfree mass. Methods Subjects (19 males, 23â81 years old; 20 females, 20â77 years old) ingested a single dose of 60 mg D3âCr and urine was collected prior to and daily for 4 days following the dose. Fasting morning urine samples was assessed for D3âCr, total Cr, D3âCrn, and total Crn concentrations, as well as isotopic enrichments of D3âCrn, by LC/MS. The 24âh urine collections over 3 days after the dose of D3âCr were also performed to determine D3âCr spillage. Total body water, fat mass, and fatâfree mass were assessed by bioelectrical impedance spectroscopy (BIS). Results Spillage of D3âCr in the urine was greater in women than men. D3âCrn enrichment and the ratio of Cr/Crn were used in an algorithm to calculate Cr pool size and muscle mass. Specifically, an algorithm was developed for the estimation of spillage based on the relationship between the fasting Cr/Crn ratio and the cumulative proportion of the D3âCr dose excreted over 3 days based on 24âh urine collections. Muscle mass corrected using the algorithm based on fasting urine levels correlated (r = 0.9967, P < 0.0001) with that corrected by measuring D3âCr dose excreted. Muscle mass measured by D3âCrn enrichment also correlated (r = 0.8579, P < 0.0001, algorithm corrected) with that measured by 24âh Crn excretion. Muscle mass measured by D3âCr dilution method correlated with intracellular water by BIS, whether using spillage corrected by the algorithm (r = 0.9041, P < 0.0001) or measured by 3 day D3âCr losses (r = 0.91, P < 0.0001) and similarly correlated with fatâfree mass by BIA (r = 0.8857 and 0.8929, P < 0.0001, respectively). Conclusions The D3âCr dilution method is further validated here as a nonâinvasive, easyâtoâuse test for measuring muscle mass. The technical issue of D3âCr spillage can be corrected for with a simple algorithm based on fasting spot urine samples. Muscle mass by Cr dilution potentially has broad applications in clinical and research settings
Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification
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 azideâalkyne 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
New astroglial injury-defined biomarkers for neurotrauma assessment
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
New astroglial injury-defined biomarkers for neurotrauma assessment
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
Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification
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 azideâalkyne
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-<i>d</i><sub>5</sub>-bromide, with reporter ions appearing at <i>m</i>/<i>z</i> 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 <i>N</i>-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
Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions
We present novel homobifunctional amine-reactive clickable
cross-linkers
(CXLs) for investigation of three-dimensional protein structures and
proteinâprotein 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