Mass spectrometry accelerates membrane protein analysis

doi:10.1016/j.tibs.2011.04.005

Trends in Biochemical Sciences

Volume 36, Issue 7, July 2011, Pages 388-396

https://doi.org/10.1016/j.tibs.2011.04.005 Get rights and content

Cellular membranes are composed of proteins and glyco- and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis, by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand–receptor pairs, and the elucidation of signaling networks originating at the PM. Here, we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.

Section snippets

Application of mass spectrometry (MS) to study membrane proteins

The water insoluble nature of transmembrane proteins renders them challenging, but not impossible, to investigate by traditional biochemical approaches in conjunction with MS [1]. In this review, we focus on the eminence of shotgun MS for accelerating the identification and study of membrane proteins. Specifically, we briefly cover recent MS advancements to determine the complete membrane proteome, as a way to better understand membrane protein topology, membrane protein–protein interactions,

Shotgun MS proteomics to determine the complete membrane proteome

The relatively low abundance of membrane proteins in unfractionated samples has undoubtedly resulted in their under-representation in large proteomic datasets. However, plasma membrane (PM) proteins are not completely absent in these datasets; they are just more challenging to identify. Several recent technological advancements, including improved sample preparation, instrumentation and better liquid chromatographic (LC) performance, have led to a substantial increase in PM protein

Proteomic methods to determine membrane protein topology

MS is one of the most powerful tools available to study proteins. MS can now easily identify and quantify proteins, determine PTMs, and also explore protein structure 29, 30. PM protein topology can be complex and is a crucial determinant of function that can be effectively investigated with MS. Membrane protein structure is notoriously difficult to study by traditional high-resolution methods such as X-ray crystallography and NMR spectroscopy. Recently, however, MS in conjunction with

Shotgun MS facilitates the mapping of membrane protein–protein interactions

MS applications are making progress toward the comprehensive identification of all PM proteins, and probe their topology. MS has also proven to be particularly useful to discover PM protein–protein interactions. Such experiments can reveal which proteins physically (and often functionally) interact, thereby representing an essential step towards elucidating the molecular function of PM proteins. Most investigations fall into one of two general approaches: isolation of membrane protein complexes

Shotgun MS identifies signaling networks across membranes

Once PM protein interaction networks have been elucidated, it is crucial to determine how they are integrated to transmit signals across PMs. The PM is a crucial cellular location for the integration of signaling events, and is enriched for surface receptors (e.g. GPCRs, receptor tyrosine kinases, adhesion signaling molecules, and channels). MS analysis of membrane protein signaling has recently gained attention and is revealing signaling pathways at unprecedented levels 60, 61. Indeed,

Concluding remarks

MS has proven to be a powerful approach to accelerate our understanding of membrane proteins by facilitating discovery-based investigations. These unexpected findings have had a significant effect on the membrane protein field and have propelled it in new and exciting directions. In summary, MS comprehensively identifies PM proteins, probes PM topology, maps PM protein–protein interactions, and elucidates PM signaling networks. The recent advancements in shotgun proteomic instrumentation and

Acknowledgments

We would like to thank Guoan Zhang, Thomas Neubert, Joris de Wit and Terunaga Nakagawa for useful discussions regarding the determination of signaling networks, and membrane protein interactions. Additionally, Albert Heck provided critical reading of the review, and Emily J. Larrimer gave valuable editorial advice. Many of the ideas presented here were developed through scientific discussion with current members of the Yates Laboratory. The authors would like to acknowledge funding support from

References (75)

L.A. Eichacker
Hiding behind hydrophobicity. Transmembrane segments in mass spectrometry
J. Biol. Chem.
(2004)
J. Moebius
The human platelet membrane proteome reveals several new potential membrane proteins
Mol. Cell. Proteomics
(2005)
B.A. Jordan
Identification and verification of novel rodent postsynaptic density proteins
Mol. Cell. Proteomics
(2004)
C.J. Nelson
A quantitative analysis of Arabidopsis plasma membrane using trypsin-catalyzed (18)O labeling
Mol. Cell. Proteomics
(2006)
X. Chen
Global topology analysis of pancreatic zymogen granule membrane proteins
Mol. Cell. Proteomics
(2008)
C.L. Han
A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease
Mol. Cell. Proteomics
(2008)
J.Y. Jia
Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling
Mol. Cell. Proteomics
(2006)
Y. Li
Enhancing identifications of lipid-embedded proteins by mass spectrometry for improved mapping of endothelial plasma membranes in vivo
Mol. Cell. Proteomics
(2009)
R.S. Boyd
Protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma
Mol. Cell. Proteomics
(2009)
B. Sun
Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics
Mol. Cell. Proteomics
(2007)

P. Hufnagel

Electrospray ionization mass spectrometry of genetically and chemically modified bacteriorhodopsins

Anal. Biochem.

(1996)

S.W. Englander

Mechanisms and uses of hydrogen exchange

Curr. Opin. Struct. Biol.

(1996)

Y. Pan

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

J. Am. Soc. Mass Spectrom.

(2010)

W. Zhang

A transmembrane accessory subunit that modulates kainate-type glutamate receptors

Neuron

(2009)

A.O. Helbig

Exploring the membrane proteome--challenges and analytical strategies

J. Proteomics

(2010)

H. Schagger

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis

Anal. Biochem.

(1994)

J. de Wit

LRRTM2 interacts with neurexin1 and regulates excitatory synapse formation

Neuron

(2009)

J. Ko

LRRTM2 functions as a neurexin ligand in promoting excitatory synapse formation

Neuron

(2009)

P.H. Huang et al.

Phosphoproteomics: unraveling the signaling web

Mol. Cell

(2008)

J.V. Olsen

Global, in vivo, and site-specific phosphorylation dynamics in signaling networks

Cell

(2006)

D.S. Spellman

Stable isotopic labeling by amino acids in cultured primary neurons: application to brain-derived neurotrophic factor-dependent phosphotyrosine-associated signaling

Mol. Cell. Proteomics

(2008)

C. Choudhary

Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes

Mol. Cell

(2009)

L. Anderson et al.

Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins

Mol. Cell. Proteomics

(2006)

A.E. Speers et al.

Proteomics of integral membrane proteins—theory and application

Chem. Rev.

(2007)

A.L. Hopkins et al.

The druggable genome

Nat. Rev. Drug Discov.

(2002)

A.B. Weinglass

Integrating mass spectrometry into membrane protein drug discovery

Curr. Opin. Drug Discov. Dev.

(2004)

C.C. Wu et al.

The application of mass spectrometry to membrane proteomics

Nat. Biotechnol.

(2003)

B. Alberts

Molecular Biology of the Cell

(2002)

I. Bethani

Spatial organization of transmembrane receptor signalling

EMBO J.

(2010)

A.E. Speers

Shotgun analysis of integral membrane proteins facilitated by elevated temperature

Anal. Chem.

(2007)

E.I. Chen

Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

J. Proteome Res.

(2007)

E.I. Chen

Comparisons of mass spectrometry compatible surfactants for global analysis of the mammalian brain proteome

Anal. Chem.

(2008)

M.P. Washburn

Large-scale analysis of the yeast proteome by multidimensional protein identification technology

Nat. Biotechnol.

(2001)

R.S. Walikonis

Identification of proteins in the postsynaptic density fraction by mass spectrometry

J. Neurosci.

(2000)

T.J. Stevens et al.

Do more complex organisms have a greater proportion of membrane proteins in their genomes?

Proteins

(2000)

N. Gupta

Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics

Nat. Immunol.

(2006)

F.Y. Che

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Mol. Cell. Proteomics

(2011)

Cited by (77)

Retrograde signaling in plants: A critical review focusing on the GUN pathway and beyond
2023, Plant Communications
Plastids communicate their developmental and physiological status to the nucleus via retrograde signaling, allowing nuclear gene expression to be adjusted appropriately. Signaling during plastid biogenesis and responses of mature chloroplasts to environmental changes are designated “biogenic” and “operational” controls, respectively. A prominent example of the investigation of biogenic signaling is the screen for gun (genomes uncoupled) mutants. Although the first five gun mutants were identified 30 years ago, the functions of GUN proteins in retrograde signaling remain controversial, and that of GUN1 is hotly disputed. Here, we provide background information and critically discuss recently proposed concepts that address GUN-related signaling and some novel gun mutants. Moreover, considering heme as a candidate in retrograde signaling, we revisit the spatial organization of heme biosynthesis and export from plastids. Although this review focuses on GUN pathways, we also highlight recent progress in the identification and elucidation of chloroplast-derived signals that regulate the acclimation response in green algae and plants. Here, stress-induced accumulation of unfolded/misassembled chloroplast proteins evokes a chloroplast-specific unfolded protein response, which leads to changes in the expression levels of nucleus-encoded chaperones and proteases to restore plastid protein homeostasis. We also address the importance of chloroplast-derived signals for activation of flavonoid biosynthesis leading to production of anthocyanins during stress acclimation through sucrose non-fermenting 1-related protein kinase 1. Finally, a framework for identification and quantification of intercompartmental signaling cascades at the proteomic and metabolomic levels is provided, and we discuss future directions of dissection of organelle-nucleus communication.
Mass Spectrometry Untangles Plant Membrane Protein Signaling Networks
2020, Trends in Plant Science
Plasma membranes (PMs) act as primary cellular checkpoints for sensing signals and controlling solute transport. Membrane proteins communicate with intracellular processes through protein interaction networks. Deciphering these signaling networks provides crucial information for elucidating in vivo cellular regulation. Large-scale proteomics enables system-wide characterization of the membrane proteome, identification of ligand–receptor pairs, and elucidation of signals originating at membranes. In this review we assess recent progress in the development of mass spectrometry (MS)-based proteomic pipelines for determining membrane signaling pathways. We focus in particular on current techniques for the analysis of membrane protein phosphorylation and interaction, and how these proteins may be connected to downstream changes in gene expression, metabolism, and physiology.
Controlled assembly of AIEgens based on a super-quadruplex scaffold for detection of plasma membrane proteins
2020, Analytica Chimica Acta
Quantification of plasma membrane proteins (PMPs) is crucial for understanding the fundamentals of cellular signaling systems and their related diseases. In this work, a super-quadruplex scaffold was designed to regulate assembly of oligonucleotide-grafted AIEgens for detection of PMPs. The nonfluorescence oligonucleotide-grafted AIEgen (Oligo-AIEgen) was firstly synthesized by attaching the AIEgen to 3′-terminus of the oligonucleotide through click chemistry. Meanwhile, the tetramolecular hairpin-conjugated super-quadruplex (THP-G4) as cleavage element and signal enhancement scaffold composited of three elements: a substrate sequence of DNAzyme in the loop region, partial hybridization region in the stem, and six guanine nucleotides to form G-quadruplex. Once the DNAzyme was anchored on the specific PMPs through aptamer-protein recognition, the substrate sequence on the loop of THP-G4 was cleaved by DNAzyme with the aid of cofactor Mn^II, resulting in the conformation switch of THP-G4 to the activated G-quadruplex scaffold. The latter could assemble Oligo-AIEgens to generate aggregation-induced emission (AIE) enhancement, resulting in a simple and sensitive strategy for detection of membrane proteins. Moreover, the DNAzyme continuously cut the next THP-G4 to achieve recycling amplification. Under the optimized conditions, this AIE-based strategy exhibited good linear relationship with the logarithm of MUC1 concentration from 0.01 to 10 μg mL⁻¹ with the limit of detection down to 4.3 ng mL⁻¹. The G4-assembled AIEgens provides a universal platform for detecting various biomolecules and a proof-of concept for AIE biosensing.
Surfaceome nanoscale organization and extracellular interaction networks
2019, Current Opinion in Chemical Biology
Citation Excerpt :
Technologies specially designed to probe the extracellular ligand space in a discovery-driven manner are AVEXIS and the ligand-receptor capture (LRC) technologies [20–22]. Affinity-purification coupled to mass spectrometry (AP-MS) methods, usually employed in the context of intracellular interactions, can in principle be also applied to cell surface proteins, by affinity tagging the cytoplasmic domains [23,24,25,26•]. However, AP-MS methods typically utilize detergents for disrupting the membrane and isolating surfaceome complexes, a step that may disrupt transient or lipid-mediated interactions of surfaceome complexes.
The reductionist view of ‘one target-one drug’ has fueled the development of therapeutic agents to treat human disease. However, many compounds that have efficacy in vitro are inactive in complex in vivo systems. It has become clear that a molecular understanding of signaling networks is needed to address disease phenotypes in the human body. Protein signaling networks function at the molecular level through information transfer via protein-protein interactions. Cell surface exposed proteins, termed the surfaceome, are the gatekeepers between the intra- and extracellular signaling networks, translating extracellular cues into intracellular responses and vice versa. As 66% of drugs in the DrugBank target the surfaceome, these proteins are a key source for potential diagnostic and therapeutic agents. In this review article, we will discuss current knowledge about the spatial organization and molecular interactions of the surfaceome and provide a perspective on the technologies available for studying the extracellular surfaceome interaction network.
Cell surface protein enrichment for biomarker and drug target discovery using mass spectrometry-based proteomics
2019, Proteomic and Metabolomic Approaches to Biomarker Discovery
Cell surface proteins play a critical role in cell-cell interactions, nutrients and metabolite transport as well as translation of extracellular signals into intracellular responses and vice versa. Due to its accessibility, the cell surfaceome represents a major biomarker and drug target resource. Importantly, ~ 70% of contemporary pharmaceuticals target cell surface proteins. Therefore, mass spectrometry (MS)-based bottom-up proteomic profiling of proteins exposed at the cell surface is increasingly used in biomarker and drug target discovery/research. However, cell surface proteins are among the most difficult to prepare and analyze. Primary obstacles are (i) poor solubility of cell surface proteins in pure aqueous buffers, (ii) high degree of heterogeneous posttranslational modifications, and (iii) exceedingly wide expression level. Therefore, enrichment of cell surface proteins prior to MS analysis is challenging and complex procedure. The purpose of this chapter is to provide an overview of common enrichment techniques for bottom-up antibody-free MS-based cell surface proteomics in search for disease biomarkers and drug targets, including Ras oncogene-driven cancers.
A high sensitive and contaminant tolerant matrix for facile detection of membrane proteins by matrix-assisted laser desorption/ionization mass spectrometry
2018, Analytica Chimica Acta
Despite the significance of membrane proteins (MPs) in biological system is indisputable, their specific natures make them notoriously difficult to be analyzed. Particularly, the widely used Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) prefers analyses of hydrophilic cytosolic proteins and has a limited ionization efficiency towards hydrophobic MPs. Herein, a hydrophobic compound (E)-propyl α-Cyano-4-Hydroxyl Cinnamylate (CHCA-C3), a propyl-esterified derivative of α-cyano-4-hydroxycinnamic acid (CHCA), was applied as a contaminant tolerant matrix for high sensitivity MALDI-MS analyses of MPs. With CHCA-C3, the detection limits of hydrophobic peptides were 10- to 100-fold better than those using CHCA. Furthermore, high quality of spectra could be achieved in the presence of high concentration of chaotropes, salts and detergents, as well as human urinary and serum environment. Also, CHCA-C3 could generate uniform sample distribution even in the presence of contaminants. This high contaminant-resistance was revealed to be ascribed to the enhanced hydrophobicity of CHCA-C3 with a lower affinity towards hydrophilic contaminants. The application of CHCA-C3 is further demonstrated by the analysis of trypsin/CNBr digests of bacteriorhodopsin containing seven transmembrane domains (TMDs), which dramatically increased numbers of identified hydrophobic peptides in TMDs and sequence coverage (∼100%). Besides, a combined method by using CHCA-C3 with fluoride solvent and a patterned paraffin plate was established for analysis of integral MPs. We achieved a low detection limit of 10 fmol for integral bacteriorhodopsin, which could not be detected using traditional matrices such as 3,5-dimethoxy-4-hydroxycinamic acid, 2,5-dihydroxyacetophenone even at sample concentration of 10 pmol.

View all citing articles on Scopus

View full text

Trends in Biochemical Sciences

ReviewMass spectrometry accelerates membrane protein analysis

Section snippets

Application of mass spectrometry (MS) to study membrane proteins

Shotgun MS proteomics to determine the complete membrane proteome

Proteomic methods to determine membrane protein topology

Shotgun MS facilitates the mapping of membrane protein–protein interactions

Shotgun MS identifies signaling networks across membranes

Concluding remarks

Acknowledgments

J. Biol. Chem.

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Anal. Biochem.

Curr. Opin. Struct. Biol.

J. Am. Soc. Mass Spectrom.

Neuron

J. Proteomics

Anal. Biochem.

Neuron

Neuron

Mol. Cell

Cell

Mol. Cell. Proteomics

Mol. Cell

Mol. Cell. Proteomics

Proteomics of integral membrane proteins—theory and application

Chem. Rev.

The druggable genome

Nat. Rev. Drug Discov.

Integrating mass spectrometry into membrane protein drug discovery

Curr. Opin. Drug Discov. Dev.

The application of mass spectrometry to membrane proteomics

Nat. Biotechnol.

Molecular Biology of the Cell

Spatial organization of transmembrane receptor signalling

EMBO J.

Shotgun analysis of integral membrane proteins facilitated by elevated temperature

Anal. Chem.

Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

J. Proteome Res.

Comparisons of mass spectrometry compatible surfactants for global analysis of the mammalian brain proteome

Anal. Chem.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology

Nat. Biotechnol.

Identification of proteins in the postsynaptic density fraction by mass spectrometry

J. Neurosci.

Do more complex organisms have a greater proportion of membrane proteins in their genomes?

Proteins

Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics

Nat. Immunol.

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Mol. Cell. Proteomics

Review
Mass spectrometry accelerates membrane protein analysis