Electron capture dissociation (ECD) [1] -an experiment generally performed within the high magnetic field of a Fourier transform ion cyclotron resonance mass spectrometerresults from the mutual storage of thermal electrons with multiply protonated peptide cations. The technique is particularly useful, as it generates random backbone cleavage with little regard to the presence of posttranslational modifications (PTMs), amino acid composition, or peptide length. Electron transfer dissociation (ETD), [2] the ion-ion analogue of ECD, is conducted in radio-frequency (RF) quadrupole ion trap devices, in which radical anions serve as electron donors. Since it can be implemented on virtually any mass spectrometer with an RF ion transfer or storage device, ETD has become an increasingly widespread dissociation method.The capture of an electron can trigger a free-radicaldriven rearrangement that results in N À C a backbone cleavage and the production of c-and zC-type fragment ions. Sometimes, however, the precursor cation captures the electron and forms a long-lived, charge-reduced species that does not separate (an ECnoD or ETnoD product).[3] This phenomenon becomes more probable as the mass-to-charge (m/z) ratio of the precursor increases. As the charge density decreases, the magnitude of intramolecular noncovalent interactions increases, so that the newly formed c-and zC-type fragment ions often remain bound following electron capture and cleavage-an obstacle of higher consequence for ETD, which is conducted under conditions of elevated pressure.[4] McLafferty and co-workers reported that photon bombardment of the precursor cation prior to ECD (activated-ion ECD, AI ECD) decreased nondissociative electron capture, [5] presumably by destroying the secondary structure of the peptide cation prior to electron capture.ETD is conducted at pressures that are approximately 10 6 times higher than those used in ECD (which is carried out at approximately 0.13 Pa). Therefore, precursor cations undergoing ETD are considerably cooler, and preactivation either with photons or through collisions is expected to produce only short-lived (< 1 ms) unfolding. Recently, we examined the use of collisions to coerce the ETnoD products into dissociating through a technique coined ETcaD (ETD in conjunction with collisional activation).[3] The method increased the number and intensity of N À C a backbone cleavages; however, the majority of the newly formed fragment ions displayed evidence of hydrogen-atom rearrangement to produce evenelectron z-type fragments and odd-electron cC-type products. ECD practitioners propose that such rearrangements occur because the c-and zC-type fragment ions are held in close proximity, so that an H atom can be abstracted from the ctype and directed to the zC-type product (this hydrogen-atom transfer occurs prior to the separation of the two fragment ions). [6,7] For large-scale sequencing applications, these rearrangements are problematic, as the mass window needed to define a possible fragment becomes too large.We ...
The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.proteomics | posttranslational regulation O CT4 is a homeobox transcription factor that was first identified for its essential role in early mammalian development (1-3). It is expressed in totipotent and pluripotent cells and down-regulated on differentiation (4-6). OCT4 is required to maintain pluripotency both in vivo and in cell culture (2,7,8), and it is indispensable for transcription factor-mediated reprogramming (9-12). Together with NANOG and SOX2, OCT4 carries out these functions by activating transcription of genes that support pluripotency and repressing genes involved in development (13)(14)(15)(16)(17).A variety of proteins, including NANOG, SOX2, and OCT4 itself, form an intricate regulatory loop that balances OCT4 expression (15,(18)(19)(20). Genomic and epigenetic studies have identified additional mechanisms that directly or indirectly influence OCT4 expression, providing a detailed view of its transcriptional regulation (21-26). OCT4 function is closely tied to its regulation, because decreasing OCT4 mRNA levels cause differentiation into trophoblast, and increasing OCT4 expression by as little as 1.5-fold causes differentiation to primitive endoderm (7). The relative stoichiometry of OCT4 and SOX2 is also important for establishing pluripotency, because the efficiency of reprogramming is dependent on the proportion of OCT4 and SOX2 transcripts (9). Thus, precise regulation of OCT4 is essential for pluripotency.Although OCT4 transcriptional regulation has been extensively studied, far less is known about its posttranslational regulation. Previous studies have speculated that phosphorylation controls OCT4 activity (27,28). For example, differences in electrophoretic mobility suggested that the homeobox domain of OCT4 is differentially phosphorylated when expressed in 293 cells compared with HeLa cells (27). Intriguingly, these different states correlated with OCT4's ability to acti...
Using a modified ETD-enabled QLT mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 SCX fractions of a LysC digest of human cells (HS) using ETD, CAD, and AI-ETD, we find that AI-ETD generates 13,405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7,968) and CAD (10,904). We also analyze 12 SCX fractions of a tryptic HS digest and find that ETD produces 6,234 PSMs, 130 PSMs, and CAD 15,209 PSMs. Compared to ETcaD, AI-ETD generates ~80% more PSMs for tryptic whole cell lysate and 50% more PSMs for LysC whole cell lysate.
Dissociations of z4 ions from pentapeptides AAXAR, where X = H, Y, F, W, and V, produce dominant z2 ions that account for >50% of the fragment ion intensity. The dissociation has been studied in detail by experiment and theory and found to involve several isomerization and bond-breaking steps. Isomerizations in z4 ions proceed by amide transcis rotations followed by radical-induced transfer of a β-hydrogen atom from the side chain, forming stable Cβ radical intermediates. These undergo rate-determining cleavage of the Cα—CO bond at the X residue followed by loss of the neutral AX fragment, forming x2 intermediates. The latter were detected by energy-resolved resonant excitation collision-activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments. The x2 intermediates undergo facile loss of HNCO to form z2 fragment ions, as also confirmed by energy-resolved CAD and IRMPD MS4 experiments. The loss of HNCO from the x2 ion from AAHWR is kinetically hampered by the Trp residue that traps the OCNH radical group in a cyclic intermediate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.