Divide and Conquer: The Application of Organelle Proteomics to Heart Failure

This Review is part of a new thematic series on Proteomics: New Methods, Biological Insights, and ClinicalPerspectives, which includes the following articles:

Overview: The Maturing of Proteomics in Cardiovascular Research [Circ Res. 2011;108:490–498]Proteomics: A Reality-Check for Putative Stem Cells [Circ Res. 2011;108;499–511]

Divide and Conquer: The Application of Organelle Proteomics to Heart Failure

New Biomarkers in Cardiovascular Disease: Potential Impact and Strategies

for Discovery The Future of Posttranslational Modifications:

From the Simple to the Complex

Jennifer E. Van Eyk, Guest Editor

Divide and ConquerThe Application of Organelle Proteomics to Heart Failure

Giulio Agnetti,* Cathrine Husberg,* Jennifer E. Van Eyk

Abstract: Chronic heart failure is a worldwide cause of mortality and morbidity and is the final outcome of anumber of different etiologies. This reflects both the complexity of the disease and our incomplete understandingof its underlying molecular mechanisms. One experimental approach to address this is to study subcellularorganelles and how their functions are activated and synchronized under physiological and pathologicalconditions. In this review, we discuss the application of proteomic technologies to organelles and how this hasdeepened our perception of the cellular proteome and its alterations with heart failure. The use of proteomics tomonitor protein quantity and posttranslational modifications has revealed a highly intricate and sophisticatedlevel of protein regulation. Posttranslational modifications have the potential to regulate organelle function andinterplay most likely by targeting both structural and signaling proteins throughout the cell, ultimatelycoordinating their responses. The potentials and limitations of existing proteomic technologies are also discussedemphasizing that the development of novel methods will enhance our ability to further investigate organelles anddecode intracellular communication. (Circ Res. 2011;108:512-526.)

Key Words: proteomics � heart failure � posttranslational modifications � organelle � communication

Chronic heart failure (HF) can be the outcome of severaldisease states such as hypertension, ischemic heart

disease, or valvular defects.1 Despite these diverse etiologies,a number of features are frequently shared, including im-paired Ca2� handling, depressed contractility, and alteredenergy metabolism. Each feature reflects functional changesin specific subcellular organelles such as sarcoplasmic retic-

ulum (SR), sarcomeres, and mitochondria.2–4 Other commonfeatures of HF (such as the activation of intracellular signal-ing in response to increased adrenergic and neurohormonalstimulation and perturbation of the spatial distribution oforganelles) also imply an orchestrated action involving mul-tiple organelles.5–7 Yet it is unclear exactly how the variousorganelles coordinate their responses under both physiologi-

Original received September 8, 2010; revision received November 9, 2010; accepted November 19, 2010. In November 2010, the average time fromsubmission to first decision for all original research papers submitted to Circulation Research was 13.2 days.

From the Johns Hopkins Bayview Proteomics Center (G.A., C.H., J.E.V.E.), Johns Hopkins University, Baltimore, MD; Istituto Nazionale per leRicerche Cardiovascolari (G.A.), Department of Biochemistry, University of Bologna, Italy; and Institute for Experimental Medical Research (C.H.), OsloUniversity Hospital–Ullevaal, Norway.

*Both authors contributed equally to this work.Correspondence to Jennifer E. Van Eyk, Mason F. Lord Bldg, Center Tower, Room 607, Johns Hopkins Bayview Proteomic Center, John Hopkins

University, Baltimore, MD 21224. E-mail [email protected]© 2011 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.226910

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cal and pathological conditions. Indeed, their dyscoordinationcould be considered another feature of the failing heart.8

Organellar interplay can occur via 2 routes: second mes-sengers or structural connections (Figure 1). The concentra-tion of second messengers (eg, Ca2�, NO�, and cAMP)fluctuates throughout the cell. It is their local levels thatregulate the activity of downstream protein targets, eitherdirectly or indirectly, via signaling cascades, and a commonfinal outcome is the posttranslational modification (PTM) oftheir protein targets. These proteins can be localized todifferent organelles, providing a means for widespread cel-lular action. A good example is Ca2�, which binds directly toboth the sarco-/endoplasmic reticulum calcium-ATPase(SERCA)2 in the SR and troponin (Tn)C in the sarco-meres.9,10 Another example is cAMP, which can activateprotein kinase (PK)A, resulting in phosphorylation of pro-teins throughout the cardiomyocyte.11

Direct physical connection is the other mechanism for or-ganelle interplay, occurring through the 3 major components ofthe cytoskeleton: microfilaments (actin), intermediate filaments(desmin, which is specific to cardiomyocytes), and microtubules(tubulin).12 The roles of microtubules and the actin cytoskeletonin cellular plasticity, motility, and protein trafficking are wellknown,13,14 as is the role of intermediate filaments in maintain-ing proper localization of different organelles.15 However, novelfunctions for modified cytoskeletal proteins in diverse cellularfunctions are emerging.16–19

We postulate that PTMs are important in the developmentof HF because they likely mediate, and therefore regulate,organellar crosstalk. To test this hypothesis, organelles can beanalyzed after subcellular fractionation using a variety ofproteomic technologies. An additional advantage of thisapproach is the enrichment of low abundant, organelle-specific proteins. The integrated analysis of the data gener-ated from several organelles can help highlight commonmolecular components involved in the coordination amongdifferent cell compartments.

In the present review, we describe how our knowledge ofHF has benefitted from the ability of proteomics to scrutinizebiological processes and reveal the new levels of complexityintroduced by PTMs. We have also included novel analyticapproaches and discuss how these can be applied to specificbiological questions regarding HF. These hands-on examplesillustrate the contribution of proteomic approaches in movingthe field forward.

Proteomics: When, Why, and HowHistorically, proteomics originated when separation technol-ogies such as 2D gel electrophoresis (2DE) and liquidchromatography (LC) were combined with mass spectrome-try (MS). The need to increase our understanding of thePROTEin complement of the genOME,” or the PROTEOME,has driven the continuous development of these technologies.With the implementation and integration of various techno-logical platforms it has become possible to separate, identify,characterize, and quantify complex mixtures comprising hun-dreds to thousands of proteins or peptides.20 These are keyfeatures to gather a more thorough understanding of intricateand elusive diseases such as heart failure. The Table gives an

overview of the most common technologies, with theiradvantages, limitations, and possible biological applications.

A few key concepts have emerged from the proteomicstudies performed over the last 16 years. First, even a singleprotein can be a complex mixture of its many modified forms.Through the specific measurement of a number of PTMs, andthe determination of the actual modified amino acid residues,it has become clear that a protein can have multiple PTMsat multiple amino acid residues. Furthermore, differentPTMs may compete with each other for a particularresidue.21 To date, more than 400 different PTMs havebeen described, although far fewer have been documented inhigher organisms such as mammals (see a detailed list athttp://www.uniprot.org/docs/ptmlist). In the cardiac subpro-teome, phosphorylation is by far the best-described PTM, andmost modified proteins have multiple phosphorylation sites(Figure 2A; Table I in the Online Data Supplement, availableat http://circres.ahajournals.org). To date, phosphorylationhas been observed for 78%, 80%, and 48% of cardiac proteinslocated in the SR, myofilaments, and mitochondria, respec-tively (Figure 2B; Online Table II). However, the majority ofthe potential PTMs have not been fully investigated, in partbecause of a lack of technologies that specifically target them.Moreover, as a cautionary note, because the extent of PTMsoccurrence is not clear and PTMs can modify the antigenicproperties of proteins, quantification methods that rely solelyon antibodies may lead to biased conclusions.

Second, in multicellular organisms, different cell typesshare many of the same proteins (proteome redundancy).These common proteins are part of essential cellular systems

Non-standard Abbreviations and Acronyms

1DE 1D electrophoresis

2DE 2D electrophoresis

BNE blue native electrophoresis

HF heart failure

IEF isoelectrofocusing

IF intermediate filament

iTRAQ isobaric tag for relative and absolute quantification

LC liquid chromatography

MRM multiple reaction monitoring

MS2 tandem mass spectrometry

O-GlcNAc O-linked �N-acetylglucosamine

Q quadrupole

pI isoelectric point

PK protein kinase

PLB phospholamban

PTM posttranslational modification

SERCA sarco/endoplasmic reticulum calcium-ATPase

SILAC stable isotope labeling with amino acid in cell culture

SNO S-nitrosothiol

SR sarcoplasmic reticulum

SRM single/selective reactive monitoring

Tn troponin

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and may reflect the minimum number of functional proteinsrequired for cell survival. This implies that the uniqueattributes of a specific cell type must be derived fromvariations in protein quantity, isoform expressions (fromsplice variants or different genes), or PTMs. Furthermore, thissuggests that the regulation of proteins at the time oftranscription and translation, as well as protein complexassembly, plays major roles in determining the exact compo-sition of the proteome and thus the cellular fate. Data tosupport this concept arise from a number of detailed MS-based analyses, including work on mitochondria isolatedfrom different tissues (heart, kidney, liver, and brain).22,23

These studies demonstrate that at least two-thirds of mito-chondrial proteome are conserved across different tissues.Because the levels of these shared proteins greatly vary fromorgan to organ,23 it is possible that the quantity, rather thanthe type of protein within functional clusters, discriminates

among different tissues. However, protein isoforms are alsoimportant, as illustrated by the cell specificity of the Tncomplex (comprising TnT, TnC, and TnI): the differentcell-specific isoforms for each subunit constitute uniqueattributes of a cardiomyocyte. In addition, various combina-tions of the isoforms (slow and fast) differentiate cardiac andskeletal myocytes.24

Third, technological development is fundamental for sci-entific advancement. Improvements in each step of theproteomic pipeline have expanded the number and types ofproteins (eg, hydrophobic, basic, etc) that can be observed.Considerable contributions have arisen from methods thatimproved protein separation (eg, 2DE25,26) or enrichment ofPTM peptides (eg, O-linked �N-acetylglucosamine27,28

[O-GlcNAc] and phosphorylation29–31). Developments inmass spectrometers have also led to enhanced speed, sensi-tivity, and mass accuracy. When coupled with advanced

Figure 1. A proteomic strategy tostudy organellar communication. Afterorganelle fractionation, proteomic meth-ods including protein separation, quanti-fication, and identification are used tostudy the different subproteomes. Inte-grated analysis of the data generatedfrom several organelles can help high-light common molecular componentsinvolved in the coordination of differentcell compartments. D indicates desmo-some; G, gap junction; MT, mitochon-drion; MYO, myofilament; nDLC, mono-or multidimensional LC. (Illustrationelements provided by Cosmocyte/BenSmith).

Figure 2. Current knowledge of PTMsin heart proteins. A, Occurrence of dif-ferent types of PTMs in proteins anno-tated with “heart expression” in thehuman protein reference database (left,only site-specific PTMs that appeared in�15 entries are shown; for a full list, seeOnline Table I) with emphasis on phos-phorylated proteins grouped based onthe total number of phosphorylation sites(right). B, Distribution of proteins anno-tated with “heart” and subcellular local-ization in either SR, sarcomeres, or mito-chondria; the total number (left) and thenumber of those that are phosphorylated(right) are provided, as reported in theUniprot Protein Knowledgebase(UniprotKB).

514 Circulation Research February 18, 2011



bioinformatic techniques, this has resulted in reduced falsediscovery rates and improved protein identification.

Hundreds of review articles on proteomics and humandiseases have been published, but not as many address theapplication of proteomic technologies in the elucidation ofcardiac disease (see selected reviews32–37). In the next section,we describe the different techniques commonly used inorganelle proteomics, beginning with subcellular fraction-ation and following each step of the proteomic workflowthrough MS-based protein characterization.

Subcellular Fractionation for ProteomicsEach separation technique used in proteomics has a limitedcapacity regarding the number and types of proteins that canbe resolved. This technical limitation prevents the detectionof low abundant proteins and cannot always be overcome bysimply increasing the total protein load as this may paradox-ically result in protein loss (eg, by precipitation). Sensitivityand resolving power can instead be increased by focusing ona particular subproteome.35,37 This requires balancing 2 as-pects: sample purity and preservation of the PTM status ofeach protein. Importantly, regardless of the methods used, itis difficult to isolate a pure organelle preparation. It is equallydifficult to prove that a preparation is pure. This arises fromthe dilemma that a subproteome can comprise residentorganellar proteins, those with multiple cellular localizationsand contaminating proteins. Enrichment of a particular pro-tein during the preparation can help, but validation throughother techniques, such as microscopy, is ultimately requiredto confirm subcellular localization.23

When compared with traditional biochemical organellepurification methods, a major concern for proteomic studies

is keeping modifications intact during fractionation. To avoidintroducing artificial PTMs during sample processing, eitherpharmacological inhibitors (eg, phosphatases, proteases in-hibitors) or harsh denaturing conditions can be used.35,38 Onthe other hand, mild conditions are required when enzymeactivity or protein–protein interactions (eg, complexes) arebeing addressed.39 For example, our group has optimized afast and reproducible method (called IN Sequence) to frac-tionate frozen heart tissue for proteomic applications: divid-ing the cardiac proteome into separate myofilament- andcytosolic-enriched fractions.40 This method was developed todeplete the high abundant myofilament subproteome, allow-ing enhanced observation of the cytosolic proteins andpreserving the original endogenous PTM status. Althoughthis method is appropriate for proteomics, it leaves themyofilament subproteome denatured, inactive, and not ame-nable to functional studies. Additional precautions may berequired for some oxidative PTMs, where the proceduresshould be performed in the dark (eg, S-nitrosylation41,42). ForPTMs that are extremely labile (because of chemical insta-bility or activation of regulatory enzymes), the speed of theprocedure will be a factor in protocol optimization. Thedecision on which approach to use depends on the biologicalquestion and the assays that will be performed downstream ofthe proteomic pipeline (such as enzyme activity and otherfunctional assays).

Protein and Peptide Separation TechniquesElectrophoretic Methods: “Classical” and“Nonclassical” Two-DimensionalGel ElectrophoresisTwo-dimensional gel electrophoresis (2DE) has been used forprotein separation since the first proteomic study.20 The 2DE

Table. Proteomic Techniques

Name Advantages Limitations References Resolution Quantification Applications

1DE • Fast and easy• Hydrophobic proteins• Inexpensive

Low resolutionQuantification

22, 23, 48, 67, 122, 127, 128, 134, 148, 165 Good Poor D, ID

2DE • Quantitative• PTM indication• Three-plex (DIGE,�3)

• Hydrophobic proteins• Operator dependent

20, 43, 58, 72, 103, 110, 127, 135, 155, 157, 160 VeryGood

VeryGood

D, Q,ID, PTM

1DLC* • Automatized• Hydrophobic Proteins• Off/Online with MS

• Low resolution• Quantification

70, 105, 127, 129, 131, 135, 148 Good Poor D, ID

2DLC • Automatized• Off/online with MS• Sensitivity

• MS workload• Quantification

65–67, 106, 135 Good N/A D, ID, PTM

Tag-free • Fast• Good dynamic range

• Requires carefulnormalization

83 N/A Good Q

MS-tag • Multiplex (�8)• Reproducible

• Limited dynamic range 52, 53, 72, 86, 148 N/A VeryGood

Q

MRM • Absolute quantification• Customizable• Multiplex (�40)

• Expensive• Time consuming

to establish

88, 89 N/A VeryGood

Q,† PTM

D indicates creating tissue, cellular, or organelle database; Q, protein quantification; ID, protein identification; PTM, protein discovery and/or validation; N/A, notapplicable.

*Mainly reversed-phase.†relative and absolute protein quantification.Original articles cited in the text are also listed in the table; in bold are the references that used cardiac samples.




protocol most widely used today was developed in themid-1970s by O’Farrell.43 Since then, 2 notable improve-ments that greatly reduce technical variability have beendeveloped: (1) the introduction of immobilized pH gradientstrips for isoelectrofocusing (IEF)44 and (2) fluorescent pro-tein labeling.45 In fact, researchers can now reproduciblyseparate thousands of protein forms and reliably quantifyprotein changes across different samples by combining thesemethods (Table).25,36,46

In the first dimension of classical 2DE, proteins areseparated according to their isoelectric points (pI) by IEF.Some PTMs, such as phosphorylation, acetylation, deamina-tion, or alkylation can modify the pI of one protein, inducinga shift along the IEF axis.47 In the second dimension, proteinsare separated according to their size using SDS-PAGE. OtherPTMs can modify protein size and, thus, electrophoreticmobility in the second dimension; these include proteolyticcleavage, glycosylation, and ubiquitination.48 Phosphoryla-tion can also slow down electrophoretic mobility, inducing anapparent alteration in protein size, most likely because of theinterference with protein–SDS interactions.49

Recently, several “nonclassical” 2DE techniques have beenrediscovered and optimized, combining different separationmethods. One example is blue-native gel electrophoresis(BNE), where proteins are separated under nondenaturingconditions. Protein complexes can be separated by BNEfollowed by further separation under denaturing conditions,either by SDS-PAGE or classical 2DE. The initial BNE stepcontributes with additional information regarding protein–protein interactions and the composition of proteincomplexes.50

2DE has the advantage of providing a quantification methodbased on the area/volume of a protein spot after gel staining.Spot matching and differential display analysis are carried outthrough semiautomated software analysis. Fluorescent stainsand, in particular, the difference in-gel electrophoresis ap-proach45 offer a very reliable platform for quantifying changesin protein levels between different experimental conditions(eg, disease versus healthy). Moreover, the image analysisthat used to be extremely laborious can now be contracted tocompanies that perform this routinely.51 Despite the advan-tages of 2DE, a few considerations have to be mentioned.Each spot visualized can be a mixture of several proteins. Inthis case, another quantitative step is required to determinethe specific protein responsible for the difference in spotintensity.52,53 In addition, a single protein may be presented asmultiple spots, each representing different forms. Therefore, a2DE gel containing 1000 spots may only represent 300 to 400different proteins.54 These factors should be taken intoaccount when protein identity and quantity changes areassigned based on 2DE.

One physical limitation of 2DE is the poor capacity forstudying hydrophobic proteins, because anionic detergentscannot be used in the first dimension (IEF).55,56 Althoughwe57,58 and others have shown previously that is possible toseparate highly hydrophobic proteins on 2DE, alternative tech-niques such as liquid chromatography (LC) or SDS-PAGE maybe a preferable strategy. 2DE is, however, currently the only

method that provides a direct indication of many different PTMsin a single analysis.

Chromatographic Methods: One- andTwo-Dimensional LCDifferent LC techniques such as ion exchange, size exclusion,affinity, and reversed-phase, can be used for the separation ofcomplex protein and peptide mixtures based on their intrinsicproperties (ie, size, charge/pI, hydrophobicity, and protein-binding properties40,59,60). In these methods, analytes areseparated in solution according to differences in their parti-tioning between the stationary and mobile phases. The mostwidely used LC in proteomics is reversed-phase, either off- oron-line with MS. A wide selection of chromatographicmaterials for reversed-phase LC is available. The choice ofstationary phase (particle size, pore size, surface area, andchemistry of the substrate surface) and, to a lesser degree,flow rate and buffer composition, can be optimized toimprove resolution.

As with gel-based methods, the combination of differentanalytic LC strategies (multidimensional LC) increases reso-lution.59–62 2D LC can be used to separate peptides afterprotein digestion (referred to as shotgun approach),63,64 orintact proteins. The latter potentially provides additionalinformation about protein isoforms and PTM status. Similarto 2DE, LC methods do not allow for unambiguous assign-ment of quantity changes based on elution profile alone, andrequire protein labeling before MS (see below).

A specific LC technique that is worth mentioning isaffinity chromatography. Affinity chromatography is basedon reversible antigen–antibody interaction and allows proteinor protein complexes to be recovered with good purity and, insome cases, with retained biological activity.68–70 The mostcommon technical challenges with affinity chromatographyare: determining which proteins are contaminants because ofnonspecific binding; and distinguishing between the directand indirect binding partners that form a protein complex.Different factors that can contribute to nonspecific bindingare ionic or hydrophobic interactions. These can be limited bycarefully optimizing both washing and binding conditions.

The use of multiple proteomic methods that combinedifferent protein and/or peptide separation methods increasesthe overall coverage of the proteome.71,72 This is particularlyimportant when studying species that have poor or underrep-resented annotation, such as rabbit or dog.73,74 In thesespecies, isolation of a higher number of peptides is requiredfor exact protein identification and characterization. In fact,enhanced detection power increases the probability of match-ing a peptide sequence to a homolog in a more annotatedspecies.

Protein Identification and QuantificationMethods: Mass Spectrometry

Today, MS is the most common method for identification ofproteins. MS has eclipsed other techniques, such as Edman’sdegradation, because of its speed and level of detail (eg, PTMcharacterization; for historical account of MS development,see Zhou et al75).




MS instruments consist of 3 modules: an ion source, a massanalyzer, and a detector. Once ionized, particles are acceler-ated and sorted according to their mass to charge ratio (m/z)in the mass analyzer, before they are detected. Thanks to thedevelopment of soft ionization methods, such as matrix-assisted laser desorption and electrospray ionization (MALDIand ESI, respectively), it is now possible to ionize largebiological molecules including peptides or even entire pro-teins. The impact of these technologies on modern science isfundamental, and their inventors were awarded the NobelPrize for chemistry in 2002.76 Common analyzers includetime-of-flight (TOF), quadrupoles (Q), and ion traps. Each ofthese has been combined in tandem mass spectrometry(MS/MS or MS2) so that peptides can be further broken downusing collision-induced dissociation. An advantage of MS2 isthat it provides detailed chemical information about theamino acid sequence.77 Because mass accuracy is best overthe low mass range, analysis of peptides rather than intactproteins allows for superior mass detection and, conse-quently, improved amino acid sequence assignment (called a“bottom-up” analysis). This fact, along with other benefits ofpeptide-centric proteomics (which include better separation,ionization, and database searching, see above) has determinedthe larger use of bottom-up approaches. However, MS canalso be used to characterize intact, nondigested proteins, withor without prior separation (also known as a “top-down”approach).78,79 Because of the continuous improvements ofMS technologies, it is foreseeable that top-down approacheswill become increasingly popular in the future.

Several approaches based on MS have been developed forquantification. These can be done with or without adding amass tag to the peptide (tag versus label-free methods,respectively).80 Label-free methods base quantification on thesignal intensity originated in the MS (peak area) or on thenumber of times a peptide is observed.81,82 Tag methodsrequire peptide labeling with isotopic tags83 via enzymatic(eg, stable isotope labeling with amino acid in cell culture[SILAC] and 18O labeling52,84,85) or chemical derivatization(eg, isobaric tag for relative and absolute quantification[iTRAQ]).86 SILAC can only be used in proliferating cells,but chemical derivatization methods are more flexible andallow labeling of proteins extracted from tissue and bodyfluids. In chemical methods, samples are labeled using tagswith the same molecular weight (isobaric). Once labeled pep-tides are introduced in the MS,2 each tag releases a differentfragment with a specific mass that can be tracked in the lowerportion of the spectra (�100-Da m/z). The intensity of the peaksgenerated by these reporter ions is proportional to the quantity ofthe protein in the original sample. Typically, at least 4 differenttags are available, allowing multiple biological conditions to beanalyzed simultaneously (multiplexing).

A newly adapted MS technology is multiple reactionmonitoring (MRM) and its closely related method single/selective reactive monitoring (SRM) (Online Figure I).87,88

MRM is a MS2 technique that allows relative and absolutequantification of proteotypic peptides (ie, peptides that have aunique amino acid sequence specific to a particular protein orisoform) and, hence, of proteins. In short, triple quadrupole(Q3) MS can be used to select a specific peptide of interest

(including modifications or polymorphism89,90) based on itsm/z, in Q1. This parent ion is then fragmented in the Q2, andthe daughter fragments (also called transition ions) aremonitored and quantified in the Q3. The choice of thetransition ions is critical because it allows monitoring aspecific peptide, based on MS2 identification. Normally, fewtransitions need to be monitored to achieve reliable quantifi-cation (n�5). In many cases, heavy isotope-labeled peptidesare used as internal standards for absolute quantification, butrelative quantification (sample A versus B) is relativelystraightforward. The biggest advantages with MRM are theunbiased quantification and the ability to distinguish betweenPTMs and other protein forms. This capacity is often missingwhen using antibodies.88 In addition, multiplexing is straight-forward compared to ELISA and a relative large number ofpeptides can be quantified during a single run.

PTM CharacterizationAs mentioned earlier “a protein” can be a complex and highlydynamic mixture of its modified forms. Important aspects ofthe function of one protein (such as activity, localization,half-life, and ability to form complexes) are all regulated byPTMs, and thus it is crucial that they be characterized. MS, aloneor in combination with separation and/or enrichment techniques,currently represents the preferred method for PTM characteriza-tion. Most PTMs are recognizable in a MS spectrum throughspecific mass signatures. For instance, phosphorylation typicallyinduces an increase in mass of 80 Da attributable to the weightof the phosphate group. A more common observation is theoccurrence of neutral loss: the loss of a phosphate group alone(80 Da) or plus water (80�18�98 Da).91 (See reviews onphospho-proteomics.92,93)

Two main factors limit the detection of peptides containingPTMs. One is the stoichiometric relation between the modi-fied peptide and all other peptides in the samples. In otherwords, because peptide detection is a competitive process andthe amount of a peptide with a single type of modification isrelatively low, more abundant, variously unmodified, andmodified peptides are likely to be detected first. In somecases, this limitation can be overcome by enriching for thespecific modified peptides downstream of protein digestion(Figure 1). Such is the case for phosphopeptides. There are afew protocols available, based either on the affinity ofphosphopeptides for positively charged metal ions94,95 or onother LC methods; reversed-phase LC96 and hydrophilicinteraction chromatography.97 Another consideration of PTManalyses is related to the stability of the modification. Forexample, the detection of phosphopeptides by MS is chal-lenged by the lability of the phosphate bond during collision-induced dissociation. Phosphoesters are weaker than peptidebonds; therefore they tend to break during fragmentation. Forthis reason, a method that catalyzes the preferential fragmen-tation of peptide bonds with improved detection of phos-phopeptides in the MS has been developed (electron transferdissociation).91 Similar considerations can be made for otherPTMs, although phosphorylation is currently the most stud-ied.98 Until recently, a technological challenge was site-specific quantification of a given PTM. With MRM, it is now




possible to measure the relative and absolute abundance ofPTMs at each site within and across different proteins.89,90

BioinformaticsBioinformatics is integral to proteomics. In fact, bioinfor-matic tools are needed to assist almost every step of theproteomic workflow. This includes the quantitative analysisof proteins and peptides after separation, as well as the MSdata handling.99 Bioinformatic tools are also used for theprediction of protein characteristics (such as pI and MWvalues, PTM consensus sequence, etc), and several algo-rithms are publicly available (eg, www.expasy.net). Alsofreely accessible are a number of curated protein databases(see Online Table III). Although these databases are essentialfor proteomic studies, it is important to mention that they arestill under development and therefore far from being exhaus-tive. The number of proteins assigned to a specific tissue ororganelle (Figure 2) depends on the way these are annotatedin the database. Also the total number of residues modified bya given PTM in a protein (Figure 2A, right pie chart; OnlineTable I) will vary depending on the annotation criteria(Online Table I). For instance, modifications can be assignedto a given protein entry based on the consensuses sequence ofthe modifying enzyme or if the modification has beenobserved in an isoform.

Nowadays, these protein databases are routinely mined byalgorithms that can match experimental MS spectra to proteinsequences fragmented in silico, in a semiautomated fash-ion.100 Algorithms such as OMSA, Mascot, X!tandem areable to extract the numeric values of peptide masses from MSspectra and then search against protein databases to assignpeptides to a protein entry. In all cases, manual inspection ofthe spectra is warranted, especially when there is an indica-tion of a potential modification. In fact, being programmed“to retrieve” (eg, assign a peptide to a protein), thesealgorithms show an inherent bias for false-positive identifi-cations. Another issue associated with database-based iden-tification is the fact that the databases can have multipleentries for a given protein (database redundancy). This caninflate the list of assigned entries. To overcome this issue andensure single assignments, protein-clustering algorithms canbe helpful identifying sequence similarities based on a highdegree of global (across the whole protein) or local (shortprotein domains) homology. Moreover, the correct assign-ment of a particular protein isoform or taxonomy (species)often requires extensive sequence coverage. This is becausean amino acid sequence that is unique for a particularisoform/taxonomy must be assigned. On one hand, databaseredundancy and poor sequence quality can generate false-positive identifications; on the other hand, the poor annota-tion of some species (eg, dog or rabbit) can decrease theprobability of identifying a protein correctly (false-negative).These and other issues with database search were recentlyidentified by the Human Proteome Organization as the majorcause of laboratory-to-laboratory variability in reporting pro-tein identification.101 Even though databases are likely toimprove in the future along with MS performance, awarenessand education on these issues will be crucial for the genera-tion of reliable data.

Divide and Conquer: One Subproteomeat a Time

The different proteomic techniques described above, fromcellular fractionation to detailed protein characterization byMS, constitute a powerful tool in studying such a complexdisease as HF. Even deeper insight on the disease can begained by dividing the cardiomyocyte into separate fractions,and analyzing the resulting subproteomes, rather than study-ing the whole tissue. In this section, we provide examples ofhow proteomics has been successfully used to increase ourunderstanding of the role of sarcoplasmic reticulum, myofil-ament and mitochondria in relation to cardiac disease.

The Sarcoplasmic Reticulum: Control ofCalcium and BeyondThe SR organelle, appearing as an intracellular membranenetwork, serves as the main storage for intracellular Ca2� incardiomyocytes. It has been the dogma that release of Ca2�

from SR, in a Ca2�-sensitive manner, is essential andnecessary for normal cardiac function.102 Thus, the observedreduction in cardiac force in HF patients has been partlyexplained by a reduction in the amounts and activity of Ca2�

cycling proteins. Particular focus has been targeted on 2 SRproteins: SERCA and its regulator, phospholamban (PLB).SERCA is the main regulator of Ca2� uptake during cardio-myocyte relaxation, and its activity is tightly modulatedthrough interaction with PLB.9

When studying SR proteins, the initial challenges are theextensive membrane network of the organelle and its connec-tion to other cellular compartments, which complicate theisolation of a pure structure. Also, the hydrophobic propertiesand relatively low abundance of the proteins comprising theSR can make them difficult to study. A number of generalstrategies have been developed for the enrichment of mem-brane proteins and these are applicable to SR proteins. Thesemethods include fractionation by density gradients and/ordifferential centrifugation steps. To prevent precipitation ofthe hydrophobic proteins, they can be solubilized using highconcentrations of detergents. This can be followed by elec-trophoretic separation and MS identification.103,104 Anotherapproach is to avoid the gel separation step by using shotgunmethods in the presence of MS-friendly surfactants that arecompatible with in-solution enzyme digestion and MS iden-tification (eg, Rapigest105). The first study to identify SERCAusing 2DE successfully combined centrifugation enrichmentwith diheptanoyl-phosphatidylcholine solubilization.105 Al-though the report showed that it is possible to achieveconsiderable enrichment of the SR, and that the SERCAprotein can be separated by 2DE, it also illustrated theexisting challenge separating SR from other membrane or-ganelles. Most preparations contain other membrane proteins(in particular from mitochondria103,105,106), and this compli-cates the goal of a precise identification of the SR subpro-teome. Because of the ambiguous definition of the SRsubproteome, leading to a poor implementation of the term“sarcoplasmic reticulum,” the proteins from this organelle areunderrepresented in different databases (eg, the ryanodinereceptor is not annotated to the SR in the UniprotKB; Figure2B; Online Table II).




A central role for SERCA2 in cardiac disease was ques-tioned last year in a study by Andersson et al.107 This studyshowed that a cardiac-specific, inducible SERCA2 knockoutmouse survived for several weeks even with no detectiblelevels of SERCA2 protein in the cardiomyocytes (based onWestern blotting). Surprisingly, under these conditions theSR still contributed with Ca2� to the cardiomyocyte contrac-tions. This result implies either that the heart uses analternative mechanism for cycling Ca2� into the SR or that asmall, nonimmunoreactive fraction of SERCA2 is present. Asmentioned, because of the problems in obtaining pure SR, itremains debatable which other SR proteins might be upregu-lated or modified as a consequence of severe SERCA2depletion. This emphasizes the need for an accurate descrip-tion of the SR subproteome and precisely how it changes withheart disease. An alternative strategy is to specifically targetSERCA2 using affinity chromatography combined with tar-geted MS measurements (eg, MRM).

Several genetically modified mouse models targeting ex-pression of SERCA and PLB have been constructed to studythe overall biological effects on Ca2�-cycling disruption(SERCA conditional knockout107; PLB knockout and over-expression108). As expected, significant effects on cardiaccontractility attributable to altered Ca2� homeostasis arecommonly observed in these models. Interestingly, by apply-ing different proteomic approaches (eg, 2DE, gel-free, MS2),it is clear that alterations in Ca2� handling induces pheno-typic effects beyond the contractile apparatus. These includechanges in cytoskeleton protein composition, membrane re-ceptors and intracellular signaling pathways, and energymetabolism.109,110

The Sarcomeric Organelle: Power Is NothingWithout ControlSarcomeric proteins are generally thought of as the molecularmotors in the cardiomyocyte, generating force and conduct-ing cellular shortening. Briefly, Ca2� released from SR bindsto TnC, which alters the interaction between the thin (actin,tropomyosin, and troponin: consisting of TnI, TnT, and TnC)and thick (myosin comprising myosin heavy chain and 2 lightchains and myosin-binding protein C) filaments, leading tomuscle contraction.10 Recently, it has been proposed thatsarcomeres should be regarded as a cellular organelle becauseof its close involvement in structural, electric, and metabolicaspects of cardiomyocyte dynamics.111 In HF, the reductionin cardiac pump function can be related to changes in the SRbut also to sarcomeric function.10 Although numerous muta-tions in the sarcomeric proteins causing familial hypertrophicand dilated cardiomyopathies have been described,112 theimportance of PTMs for sarcomeric proteins is also wellestablished.113

In the Tn complex, TnI is the inhibitory subunit111 and itsfunction is highly regulated by phosphorylation. The impor-tance of Ser23 and Ser24 in controlling cardiac contractilityand myofilaments responsiveness to Ca2� has been exten-sively studied.114 Recently, several other phosphorylationresidues were identified, and currently there are 7 known sitesin humans (Ser23, Ser24, Ser31, Ser42, Ser44, Thr141, andSer150)115; some of these are also important in the control of

cardiac dynamics.114 In contrast, when exploring the basal invivo phosphorylation of TnI in a murine model, only theSer22/23 sites (equivalent to the human Ser23/24) were foundto be modified.116 The biological importance of these TnIphospho-sites is under debate.115 Our group has developedMRM assays to specifically quantify and monitor the phos-phorylation status of 6 of the identified TnI phospho-sites todetermine the stoichiometric ratios of each residue in vivo.

One contribution of proteomic technologies to basic sci-ence is the rapid discovery of new PTMs, including themodified residues. An example, which was recently identifiedin the sarcomeric proteins, is acetylglucosamine at Ser andThr residues (O-linked �N-acetylglucosamine [O-GlcNAc]).When first discovered, identification of O-GlcNAcylationwas challenging and time-consuming.117 However, enrich-ment methods of today, in combination with sensitive MS,have established widespread occurrence of this PTM.21 Re-cently, O-GlcNAcylation was implicated in myofilamentcontractile alterations.118 Ramirez-Correa et al discoveredmyofilament Ca2� desensitizing when exposing cardiac TX-100–skinned muscle fibers to GlcNAc (without disturbingthe phosphorylation status of Ser23/24). Using MS-basedmethods, the group identified 32 O-GlcNAcylation sites inthe myofilament subproteome, one being Ser150 of TnI.Knowing that there is extensive crosstalk betweenO-GlcNAcylation and phosphorylation,21 it will be interest-ing to see how the alternate modification at Ser150 contrib-utes to the biological function of TnI and the regulation ofmuscle contractile function.

Mitochondrial Proteomics: PTM Switches toControl Energy ProductionBecause the heart consumes more ATP than any organ of thebody, it is particularly rich in mitochondria.119 During thedevelopment of HF, both the energy demands and metabo-lism change, showing a remarkable decrease in oxidativephosphorylation and a shift toward glucose over fatty acidutilization.120 Interestingly, during HF therapy such as cardiacresynchronization, coordinated changes in the mitochondrialproteome suggest a reversal to a healthier phenotype. Thesealterations include proteins controlling metabolism and ATPproduction, establishing the importance of mitochondria inmodulating the development of HF.58

The extensive application of proteomics to mitochondriaalso has historical and practical reasons. The study of mito-chondria has exploited protein biochemistry for many de-cades, and proteomics itself can be considered an evolution ofprotein biochemistry. Furthermore, methods to enrich andpurify the mitochondria are also several decades old, repre-senting one of the most long-lived examples of proteomesubfractionation.121 Proteomic studies have certainly in-creased our understanding of mitochondria by providingevidence for the large and unexpected occurrence of PTMs.

The human mitochondrial proteome was originally charac-terized by Taylor et al, who initially described 651 mitochon-drial and mitochondrial-associated proteins.122 Now, curateddatabases for mitochondrial proteins are also available,123

including one recently compiled by Mootha and colleagues,who assigned 1098 mitochondrial proteins (MitoCharta) by




combining extensive MS characterization with microscopyand computation to confirm mitochondrial protein localiza-tion.23 Unfortunately, there is a lack of equivalent databasesfor the less annotated species that are used in cardiac research(eg, rat, canine, porcine). Moreover, according to Forneret al,22 who recently compared the proteome of mitochondriaextracted from different rat organs (skeletal muscle, heart andliver), mitochondrial proteins can be annotated for otherorganelles (but not mitochondria) in widely used databases.The authors also pointed out that mitochondrial proteomesfrom different organs are similar, at least qualitatively, withone-third of the proteins identified being expressed exclu-sively in one organ. However, what seem to make a func-tional difference are the levels of expression of certainproteins.

Because of the high complexity of the mitochondrialproteome, and its inclusion of proteins with highly diverseproperties, the proteomic approach for its study must becarefully selected. For instance, membrane proteins, whichare more basic124 and overrepresented in the mitochondria,122

are normally poorly separated by 2DE.56 We reported previ-ously that �1000 mitochondrial protein forms, purified fromthe heart tissue in an animal model of HF, can be separatedusing 2DE.58 These include basic membrane proteins such asProhibitin 2 (pI�10.04) and voltage-dependent anion chan-nels (pI�7.48), which could be nicely resolved throughimproved sample loading using paper bridge.57 Other groupsused LC-MS2 in combination with 1DE22 or iTRAQ125 tocompare the proteins from cardiac mitochondria under dif-ferent conditions.

Targeted strategies can be used to focus on a specificportion of the mitochondria, such as the respiratory chain.The respiratory chain consists of 5 protein complexes (I to V);the first 4 generate the proton gradient that is used bycomplex V to synthesize ATP.126 At least 3 of the 5complexes in the chain have been investigated using pro-teomics.127,128 This is not surprising, because some of themcould be considered a proteome on their own. For instance,complex I is constituted by 45 subunits.129 Moreover, thiscomplexity is further increased by PTMs.130–132 Recently,Palmisano and colleagues were able to identify phosphoryla-tion sites in at least 5 subunits of complex I by combiningBNE and phosphopeptide enrichment through titanium diox-ide.35,130 We demonstrated complex V (ATP-synthase) phos-phorylation and its effects on ATP synthesis using, respec-tively, phosphopeptide enrichment and mutation analysiscombined with functional assays.58,133 Recently, Ping et alidentified 61 phosphorylation sites in all major mitochondrialpathways, including 10 for the electron transport chain and 4for complex V, in the murine heart.134

Other mitochondrial compartments can be enriched to peerdeeper and deeper into this proteome and its modifications(Figure 3).135–137 The very notion that mitochondrial proteinscan be modified is a fairly recent advent, and it opens the wayto a more accurate description of the regulation of energyproduction in the normal and failing hearts. PTMs, amongothers, govern the way proteins interact to form supramolecu-lar complexes, and their characterization is crucial.

Subproteomics and Cellular Interplay: PuttingHumpty Dumpty Back Together

As described so far, isolating and studying single organellesis a good approach to uncover substantial information regard-ing their regulation and interplay. However, organelles can betied together by either oscillations in the levels of secondmessengers or physically, through structural proteins. In theheart, perturbation of the complex and coordinated interplaybetween different cellular compartments might be an under-lying sign of disease.

Second Messengers and Proteomics: Identifyingthe TargetsIn this section, we discuss the involvement of the secondmessengers Ca2�, NO�, cGMP, and cAMP in organellecommunication in relation to HF. In healthy cardiomyocytes,contraction and relaxation are triggered by a transient rise anddecline in the concentration of intracellular Ca2�, whichcycles among sarcolemma, SR, and sarcomeres.10 Ca2�

enters the cell through ion channels in the sarcolemma,triggering further release of Ca2� from the SR. This occurswhere Ca2� channels in the plasma membrane and SRmembrane are in close proximity. A common feature in HF isthat this proximity becomes disorganized, which inhibitsefficient SR-Ca2� release and results in weakened cardiaccontractions.7 It has also become evident that mitochondriacan provide a significant storage of Ca2� and that mitochon-drial Ca2� uptake increases ATP production.138 Thus, on abeat-to-beat basis, Ca2� both induces muscle contractionsand increases ATP production to meet the needs of height-ened energy demand. This supports the idea that Ca2� is animportant factor in organellar interplay (Figure 4). Ca2� canalso be stored intracellularly in microdomains, where, unlikein the cytosol, levels do not fluctuate during the actionpotential.139 This pool of Ca2� can bind to and activatecytosolic enzymes (eg, calmodulin and calcineurin), therebyinducing Ca2�-dependent signaling cascades. One importantaspect involved in cardiac disease is the prohypertrophicresponse mediated by the Ca2�/calcineurin-dependent signal-ing pathway.140

In contrast to Ca2�, NO� diffuses across the sarcolemma.NO� can directly alter protein function by covalently modi-fying cysteine thiols to generate S-nitrosothiol (SNO). Re-cently, Lima et al suggested that a majority of the NO� effectsare mediated by this PTM.141 Because of its labile nature,considerable work has been invested in the improvement ofproteomic techniques to study SNO.42 It is important todevelop robust technology for the identification and quanti-fication of SNO, because its presumed role as a regulator incardiovascular disease is growing.141 Recently, impairedS-nitrosylation of the ryanodine receptor was shown to induceCa2� leak from the SR and thus contribute to cardiacdysfunction.142

Another important feature of NO� is the induction ofanother second messenger, cGMP, through activation of thesoluble guanylyl cyclase. cGMP activates PKG, similar tocAMP activation of PKA; both induce signaling cascades,resulting in altered cardiomyocyte growth and function.11,143

Interestingly, both PKG and PKA signaling cascades con-




verge in the phosphorylation of several proteins (eg, bothenzymes specifically induce phosphorylation of Ser 23/24 inTnI115,144). With respect to organelle communication, PKAand PKG phosphorylate proteins that are located in differentcellular compartments: SR (PKA/PKG: ryanodine receptorand PLB); myofilaments (PKA/PKG: TnI; PKA: myosinbinding protein C); mitochondria (PKA/PKG: mitochondrialATP-regulated potassium channel); and sarcolemma (PKA/PKG: L-type Ca2� channel).11,145–147 Phosphorylation viathese kinase signaling cascades can be important in transmit-ting signals throughout the cell and coordinating the action ofseveral organelles. Recently, an impressive 20 443 phos-phopeptides where quantified in a single study.148 Althoughthis was done in HeLa cells, similar levels will most likelyoccur in cardiomyocytes. Other PTMs that have been suc-cessfully enriched for are acetylation, methylation, ubiquity-

lation, and glycosylation,149 but such investigation in thecardiac system remains recent.

Organelles Physical Connection: One Ring to RuleThem AllOrganelles can also communicate through the physical con-nection mediated by the cytoskeleton. For instance, the manyinteractions between intermediate filaments (IFs), and allmajor cell compartments make them strong candidates tomediate organelles communication.15 In the heart, IFs areconstituted by desmin and have been found to regulate thefunction of myofilaments,150,151 mitochondria,15 and gapjunctions.152 The central role of IFs in HF is corroborated byrecent proteomic studies that investigated all major subpro-teomes of the cardiac cell and found that modified desminforms were altered as a common denominator. The increase

Figure 3. Proteomic to study PTM reg-ulation of protein-protein interaction.Proteins and protein complexes can bealternatively separated from organelles,such as mitochondria. As an example,schematically presented, ATP-synthase(complex V) can be denatured into itsindividual subunits or separated as awhole. The first approach is more suit-able to monitor PTMs, the latter providesinformation on complex composition.The integrated analysis of the two strate-gies can indicate the role of PTMs incomplex formation. (Illustration credit:Cosmocyte/Ben Smith).

Figure 4. Organelle dyscoordination inheart failure. Schematic presentation offeatures in a failing cardiomyocyte:increased neurohormonal stimulationand mechanical stress affects cellularcontractility, energy metabolism, con-duction, intracellular signaling, and cellstructure mislocalization. We proposethat dysregulation of various organellescould be considered another feature ofthe failing heart. We further hypothesizethat this is regulated through changesin the homeostasis of second messengers,resulting in dynamic regulation of PTMs,and by the disruption of cytoskeletalstructures such as IFs. D indicates des-mosome; G, gap junction; MT, mito-chondrion; MYO, myofilament.




of desmin PTM forms with disease is a consistent observationfrom proteomic studies that investigated various human andanimal models of HF, so it is highly unlikely that itsalterations are nonspecific.58,153–162

Desmin coenriches with the myofilament fraction of theproteome because IFs are “wrapped” around the sarcomeresand keep them aligned.150 Desmin mutations cause contractilefailure, and it was recently postulated that the misalignmentof the sarcomeres that arises when desmin cytoskeleton isdisrupted may represent a cause of contractile failure itself(Figure 4).150,162 Our group has recently reported that thelevels of PTM forms of desmin are altered in the mitochon-drial fraction of a canine model of HF compared with bothshams and animals rescued by cardiac resynchronizationtherapy.58 This observation opens an interesting line ofinvestigation, because desmin is known to regulate mitochon-drial positioning and behavior through, albeit, an unknownmechanism.5,155 One potential explanation is that desmin,maintaining the spatial organization between sarcomeres andmitochondria, ensures that energy production and consump-tion are properly coupled (Figure 4). In addition, the integrityof IFs cytoskeleton plays a major role in cardiac conductionbecause gap junction localization at the intercalated discs isensured by desmin through the interaction with the desmo-somes (Figures 1 and 4).152 Lateralization of gap junctions,which is observed in the failing heart, may be a cause ofarrhythmias, and can be induced by mutation of desmin ordesmosomal proteins, at the sites of interaction with the IFs.19

Moreover, a number of novel functions have been attributedto the IFs in the heart, including the regulation of the cardiaccommitment of stem cells,16 of autophagy,17 and, morerecently, of translation.18 We believe that PTMs finely regu-late these functions of the IFs.

Quantification of desmin in human HF, using immuno-staining techniques, has generated controversial results be-cause different groups reported either an increase or theabsence of desmin in the cardiomyocytes of HF patientscompared to healthy subjects.163,164 On the other hand,desmin quantification by 2DE has shown that multiple PTMforms of the protein are increased during human HF.160 Wereported the increase of modified forms of the protein incultured cardiomyocytes that become hypertrophied withendothelin-1 treatment.155 Therefore, we postulate that thediscrepancy generated by immunodetection studies may beattributable to PTMs. This exemplifies how proteomics couldhelp in deciphering the molecular reasons for this controversyand, most importantly, the biological value of these modifi-cations. A detailed characterization and quantification ofdesmin PTM species not only would help elucidate thefunctions of the IFs in the heart but could also highlight novelcandidate biomarkers for cardiac disease.157

Several groups have reported changes in desmin-modifiedforms with disease, in vivo, and Capetanaki et al establisheda causal link between desmin modification and the formationof aggregates in a transgenic mouse model of HF.15 Theincrease of an “acidic” form of desmin by 2DE was reportedin the same study. However, despite the existence of severalreported in vitro desmin modifications, desmin PTMs havebeen characterized in detail in vivo only recently. We

identified the first phosphorylation sites of desmin in theheart and linked them to HF in both a canine model and inhumans.157 Desmin has also been found phosphorylated oracetylated in noncardiac cells and tissue109,148,165; this wasunexpected, because desmin is thought to be specificallyexpressed in myocytes. The discoveries of desmin PTMsopen new and exciting lines of investigation to address thefunctional roles of these modifications, particularly in HF.

In conclusion, the disruption of IF networks affects thefunction of at least 3 of the most important cell compartmentsin the heart and possibly regulates many other aspects of cellfunction as well. Desmin phosphorylation and proteolysishave been long-known regulators of its assembly and func-tion in vitro,166,167 yet without site information, in vivo,progress has been hampered. Further study of the biologicalrole played by these PTMs will be helpful in elucidating themechanisms that lead to IF lattice modification during HFand its effect on organellar interplay.

Conclusions and Future PerspectivesThere is a need to develop novel, experimental approaches tostudy HF. We have reviewed how proteomic studies andtechnologies have enhanced our understanding and revealednew levels of complexity in HF at the cellular, organelle, andprotein levels. Integration of data obtained from differentsubproteomes and system-wide proteomic studies is impor-tant in understanding cellular phenotypes. We have empha-sized the need to understand the complex and coordinatedinterplay between organelles. We hypothesize that the mech-anisms involved in synchronizing different cellular compart-ments may be disrupted with HF. Furthermore, we proposethat the regulation and end effectors of this communicationreside, at least in part, with the induction of PTMs of targetproteins. The quantification and characterization of proteomechanges are a necessity and will drive the development andadaption of new proteomic approaches. In this way, we willeventually understand the global communication that drivesthe cellular phenotype.

AcknowledgmentsWe thank Christopher Murray for critical comments on the manu-script and Lars Husberg for data-mining support of the humanprotein reference database.

Sources of FundingJ.E.V.E. is supported by National Heart, Lung, and Blood Institutegrants HV-10-05 (2) and P50 HL 084946-01; and the ClinicalTranslational Science Award at Johns Hopkins University. C.H. issupported by Oslo University Hospital–Ullevaal. G.A. is supportedby NIH Program Project grant P01-HL077180 and Compagnia diSan Paolo, Turin, Italy.

DisclosuresNone.

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Giulio Agnetti, Cathrine Husberg and Jennifer E. Van EykDivide and Conquer: The Application of Organelle Proteomics to Heart Failure

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unmodified modified unmodified modifiedA. f

+

f

digestion

f f

Parent CID Daughter

PROTEINS PEPTIDESB.

D

ty, cps

unmodified

unmodified

Q1 Q2 Q3

D

modified intensit

modifiedD

elution time, min

modified

Online Figure I.MRM quantification of modified and unmodified peptides by triple Q MS. (A )I iti l di ti f t i i t tid (B) Th tid i t i i j t d i th t i l d l MS hInitial digestion of proteins into peptides. (B) The peptide mixture is injected in the triple quadrupole MS, wherethe quadrupole Q1 is used to select a specific peptide (parent). The parent ion is fragmented in the Q2 andtransition (daughter) ions are monitored in the Q3. Relative quantification is obtained by comparing the spectralcount from different transitions. Normally, five to six pairs (parent and daughter) for each peptide are monitoredin order to achieve a reliable quantification. D: detector.

Michelle

Text Box

Agnetti et al., Online Supplement

Online Table I: See separate excel spreadsheet Online Table II: See separate excel spreadsheet Online Table III: Selected protein databases Database Features link

NCBI

• Merging annotations from GenBank, RefSeq, TPA, SwissProt, PIR, PRF and PDB

• The most comprehensive resource of protein sequences (ref Martens L. Methods Mol Biol. 2009)

• Heterogenic in sequence reliability, annotation, and peptide sequence redundancy (ref Martens L. Methods Mol Biol. 2009)

• Limited additional biological information • Curated by the National Center for

Biotechnology Information (NCBI) at the National Institutes of Health (NIH)

ncbi.nlm.nih.gov/protein

Uniprot Protein knowledgebase (UniprotKB)

• Including both manually (Swiss‐Prot) and automatically annotated (TrEMBL) entries

• Currently containing more than 519 300 curated entries

• No particular focus regarding species or PTMs • Extensive additional biological information • Curated by UniProt comprising the European

Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR)

www.uniprot.org

The human protein reference database (Hprd)

• Manually curated • Currently comprising more than 93 700 PTMs • Restricted to the human proteome • Curated by The Pandey lab and the Institute of

Bioinformatics at Johns Hopkins University.

www.hprd.org

PhosphositePlus®

• Manually curated • Currently containing more than 71 400 curated

entries on phosphorylation sites • Major focus on phosphorylation sites in mouse

and human • Including non published PTMs from Cell

Signaling Technology (CTS) • Curated by CTS

www.phosphosite.org

Divide and Conquer: The Application of Organelle Proteomics to Heart Failure

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