Mass spectrometry accelerates membrane protein analysis

Savas, Jeffrey N.; Stein, Benjamin D.; Wu, Christine C.; Yates, John R.

doi:10.1016/j.tibs.2011.04.005

Cited by 69 publications

(77 citation statements)

References 73 publications

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“…In-depth studies of membrane proteins have proven to be difficult because of their low abundance and hydrophobicity 1,4 . However, recent advances in proteomic technologies make it possible to investigate proteins in this cell compartment, including previously unannotated membrane proteins, to an impressive depth [4][5][6][7][8][9] .…”

Section: Resultsmentioning

confidence: 99%

“…This membrane system includes an extensive transverse-tubular structure enriched in proteins involved in excitation-contraction coupling and specialized regions abundant in lipid rafts and caveolae required for transport and signalling. Membrane proteins in these regions act as signalling receptors, enzymes, transporters, cell-cell adhesion anchors and cell-cell signal propagators 1 . Electrical current propagation throughout the heart is mediated through precisely regulated ion movements coordinated by various cell surface and gap junction proteins 2,3 .…”

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Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

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Section: Resultsmentioning

confidence: 99%

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Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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“…To ensure broad synaptic proteome coverage, we prepared barrel cortex synaptosome fractions for analysis. The reduced level of complexity of synaptosomes allows the LCLC-MS/MS method to analyze nearly all proteins present in the sample (23). We used a third group of animals, sensory-normal mice labeled by 15 N (Fig.…”

Section: Resultsmentioning

confidence: 99%

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Butko¹,

Savas

Friedman³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high-and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensorydeprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. mass spectrometric proteomics | somatosensory cortex | experience-dependent plasticity | synaptic protein dynamics S ensory information is encoded in the brain by activation of specific neuronal circuits that operate via chemical synapses. Important sensory experiences are stored by strengthening activated synapses in the circuit, a process known as "experience-dependent plasticity" (1). When animals are deprived of sensory experience during development, for example, by trimming rodent whiskers from birth to 30 d after birth, synaptic strength and mature synaptic morphology are attenuated, suggesting that the low activity in the deprived circuits interferes with normal development (2-7). However, the molecular changes that alter these synaptic properties in response to experience remain unclear. Synaptic activation has been shown to promote regulated and highly localized effects on synaptic proteins such as local translation, recruitment, and targeted degradation (8-11), and this spatiotemporal control of protein availability is required for long-term plasticity and synaptic maturation, which are fundamental in memory formation and storage and ultimately for an organism's survival (12-14). The need for highly controlled protein availability is highlighted in studies of neurological diseases, which often result from the disruption of protein availability at the synapse (15-17). ...

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“…higher concentrations of SDS and DTT) in combination with optimal enzymatic proteolysis conditions (e.g. sodium-deoxycholate; León et al, 2013), especially suitable for insoluble, multimeric, or membrane proteins (Savas et al, 2011). Moreover, PRM can be used for relative and absolute protein quantitation of target proteins present in highly complex protein backgrounds based on its high specificity and sensitivity (Domon and Aebersold, 2010;Gallien et al, 2012;Picotti and Aebersold, 2012).…”

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Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77-to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9-to 7.1-fold or 22.5-to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3-to 8-fold or 91-to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codonoptimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

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Mass spectrometry accelerates membrane protein analysis

Cited by 69 publications

References 73 publications

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

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