Electron Transfer Dissociation Research Articles

Enhanced Electron Transfer Dissociation of Peptides Modified at C-terminus with Fixed Charges

The impact of the conversion of carboxylates in peptides to basic or fixed charge sites on the outcome of electron transfer dissociation (ETD) is evaluated with respect to ETD efficiency and the number of diagnostic sequence ions. Four reagents, including benzylamine (BA), 1-benzylpiperazine (BZP), carboxymethyl trimethylammonium chloride hydrazide (GT), and (2-aminoethyl)trimethylammonium chloride hydrochloride (AETMA), were used for the carboxylate derivatization, with the first two replacing the acidic carboxylate groups with basic functionalities and the latter two introducing fixed charge sites. The ETD efficiencies and Xcorr scores were compared for both nonderivatized and derivatized tryptic and Glu-C peptides from cytochrome c. Derivatization of the carboxylate increases the average charge states, the number of fragment ions, and the dissociation efficiencies of peptides, especially for the fixed charge reagent, AETMA.

Electron Transfer Dissociation Mass Spectrometry of Hemoglobin on Clinical Samples

A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic applications.

Higher Energy Collision Dissociation (HCD) Product Ion-Triggered Electron Transfer Dissociation (ETD) Mass Spectrometry for the Analysis of N-Linked Glycoproteins

Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.

Electron transfer dissociation: Effects of cation charge state on product partitioning in ion/ion electron transfer to multiply protonated polypeptides

The effect of cation charge state on product partitioning in the gas-phase ion/ion electron transfer reactions of multiply protonated tryptic peptides, model peptides, and relatively large peptides with singly charged radical anions has been examined. In particular, partitioning into various competing channels, such as proton transfer (PT) versus electron transfer (ET), electron transfer with subsequent dissociation (ETD) versus electron transfer with no dissociation (ET,noD), and fragmentation of backbone bonds versus fragmentation of side chains, was measured quantitatively as a function of peptide charge state to allow insights to be drawn about the fundamental aspects of ion/ion reactions that lead to ETD. The ET channel increases relative to the PT channel, ETD increases relative to ET,noD, and fragmentation at backbone bonds increases relative to side-chain cleavages as cation charge state increases. The increase in ET versus PT with charge state is consistent with a Landau–Zener based curve-crossing model. An optimum charge state for ET is predicted by the model for the ground state-to-ground state reaction. However, when the population of excited product ion states is considered, it is possible that a decrease in ET efficiency as charge state increases will not be observed due to the possibility of the population of excited electronic states of the products. Several factors can contribute to the increase in ETD versus ET,noD and backbone cleavage versus side-chain losses. These factors include an increase in reaction exothermicity and charge state dependent differences in precursor and product ion structures, stabilities, and sites of protonation.

Spatial extent of fragment-ion abundances in electron transfer dissociation and electron capture dissociation mass spectrometry of peptides

Earlier work from the first author's group has suggested that, in electron capture dissociation (ECD) or electron transfer dissociation (ETD) mass spectrometry experiments, an electron is initially attached into a Rydberg orbital centered at one of the peptide's positive sites (likely a protonated N-terminus, Lysine, Arginine, or Histidine). Moreover, this earlier work predicted that only Rydberg orbitals having principal quantum numbers n=3–6 are populated in ECD and only n=3 and 4 in ETD (when an anion donor having an electron binding energy of ca. 0. 6eV is used), and that the populations of these levels are very similar in the nascent charge-reduced peptide. Based upon these predictions, the present paper develops a framework for predicting the abundances of closed-shell c and open-shell z fragments as a function of distance along the backbone from the site initially holding the attached electron in a Rydberg orbital. The framework is not aimed at differences in branching ratios caused by differences in the physical properties of side chains along the backbone but on the spatial distances between the charged site holding the electron and the backbone amide units. The predictions of this model are tested using ECD and ETD data from derived from experiments carried out using simultaneous infrared photo-activation of the parent ions. Such activated-ion (AI) experiments are thought to disrupt much of the parent ion's secondary structure, which we believe allows us to make more reliable estimates of distances between the charged sites and the various amino acids’ amide groups. The abundance patterns predicted based upon the framework described herein are found to be reasonably consistent with the experimental data. However, the data also provide evidence that internal solvation of the peptide's charged sites remains intact even under AI conditions, and that some of these solvated-ion structures (those involving a charged Lys or N-terminal amine) contribute incrementally to the abundances of fragment ions arising from cleaving nearby NCα bonds. As a result, we conclude that ECD and ETD fragment ion abundances are determined by a combination of factors: (i) internal solvation of charged sites, (ii) spatial distributions or Rydberg orbitals charge densities within several residues of charged sites, and (iii) variations induced by differences in physical properties of side chains. It is primarily the first two of these three that the present paper addresses.

Abstract 31: Regulation of Acetylation Restores Proteolytic Function in Diseased Myocardium

RATIONALE: Proteasome complexes play essential roles in maintaining cardiac protein homeostasis in normal and stressed conditions. However, proteasomal function is often compromised in diseased myocardium; and the regulation of proteasomal dynamics remains poorly understood. METHODS AND RESULTS: We studied cardiac proteolytic function and its regulation by acetylation in murine and human models of heart disease. Proteasomes, from both normal and diseased myocardium, were treated with histone de-acetylases (HDACs) inhibitors and they exhibited enhanced proteolytic capacity in vitro and in vivo. This unique regulatory paradigm was first examined in a murine model of ischemic injury (n=5); where diminished proteolytic function in the ischemic hearts was restored by HDACs inhibition (either by SAHA or by Sodium Valproate) in a dose-dependent fashion. Importantly, this phenomenon was validated in human samples, where inhibition of HDACs augmented proteolytic functions in the diseased myocardium (n=5). Using high resolution LC-MS/MS coupled with a combined collision-induced dissociation and electron-transfer dissociation approach, the acetylome (N-terminal and lysine) of cardiac 20S proteasomes was delineated. Targeted enrichment strategy of the posttranslationally modified peptides enabled the capture of eight lysine and nine N-terminal acetylation sites in the murine heart, contributing to the first comprehensive acetylome map for the cardiac 20S proteasomes. Among them, at least six lysine acetylation sites were inducible by HDACs inhibitors. Furthermore, parallel investigations using cardiac 20S proteasomes pinpointed the functional impact of HDAC inhibitions to specific acetylation sites on the 20S proteasomal subunits. CONCLUSIONS: Proteasomal biology is modulated by acetylation modifications in the heart. This regulatory mechanism is punctual and potent; and it was observed both in an acutely pathological murine model of ischemia-reperfusion injury as well as in a chronic human disease of end-stage heart failure. These findings demonstrate the utility of pharmacological interventions (e.g., HDAC inhibition) to restore damaged proteolytic function in diseased myocardium.

Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator

Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

Tearing the Top Off ‘Top-Down’ Proteomics

Tearing the Top Off ‘Top-Down’ Proteomics

Protein O-glycosylation analysis

This review provides an overview on the methods available for analysis of O-glycosylation. Three major themes are addressed: analysis of released O-glycans including different O-glycan liberation, derivatization, and detection methods; analysis of formerly O-glycosylated peptides yielding information on O-glycan attachment sites; analysis of O-glycopeptides, representing by far the most informative but also most challenging approach for O-glycan analysis. Although there are various techniques available for the identification of O-linked oligosaccharides, the focus here is on MS fragmentation techniques such as collision-induced fragmentation, electron capture dissociation, and electron transfer dissociation. Finally, the O-glycan analytical challenges that need to be met will be discussed.

Hexuronic Acid Stereochemistry Determination in Chondroitin Sulfate Glycosaminoglycan Oligosaccharides by Electron Detachment Dissociation

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated (0,2)X(3) and Y(3) ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

Perturbing Peptide Cation-Radical Electronic States by Thioxoamide Groups: Formation, Dissociations, and Energetics of Thioxopeptide Cation-Radicals

Thioxodipeptides Gly-thio-Lys (GtK), Ala-thio-Lys (AtK), and Ala-thio-Arg (AtR) in which the amide group has been modified to a thioxoamide were made into dications by electrospray ionization and converted to cation-radicals, (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•), by electron transfer dissociation (ETD) tandem mass spectrometry using fluoranthene anion-radical as an electron donor. The common and dominant dissociation of these cation-radicals was the loss of a hydrogen atom. The dissociation products were characterized by collision-induced dissociation (CID) multistage tandem mass spectrometry up to CID-MS(5). The ground electronic states of several (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•) conformers were explored by extensive ab initio and density functional theory calculations of the potential energy surface. In silico electron transfer to the precursor dications, (GtK + 2H)(2+), (AtK + 2H)(2+), and (AtR + 2H)(2+), formed zwitterionic intermediates containing thioenol anion-radical and ammonium cation groups that were local energy minima on the potential energy surface of the ground electronic state. The zwitterions underwent facile isomerization by N-terminal ammonium proton migration to the thioenol anion-radical group forming aminothioketyl intermediates. Combined potential energy mapping and RRKM calculations of dissociation rate constants identified N-C(α) bond cleavages as the most favorable dissociation pathways, in a stark contrast to the experimental results. This discrepancy is interpreted as being due to the population upon electron transfer of low-lying excited electronic states that promote loss of hydrogen atoms. For (GtK + 2H)(+•), these excited states were characterized by time-dependent density functional theory as A-C states that had large components of Rydberg-like 3s molecular orbitals at the N-terminal and lysine ammonium groups that are conducive to hydrogen atom loss.

Characterization of O-Sulfopeptides by Negative Ion Mode Tandem Mass Spectrometry: Superior Performance of Negative Ion Electron Capture Dissociation

Positive ion mode collision-activated dissociation tandem mass spectrometry (CAD MS/MS) of O-sulfopeptides precludes determination of sulfonated sites due to facile proton-driven loss of the highly labile sulfonate groups. A previously proposed method for localizing peptide and protein O-sulfonation involves derivatization of nonsulfonated tyrosines followed by positive ion CAD MS/MS of the corresponding modified sulfopeptides for diagnostic sulfonate loss. This indirect method relies upon specific and complete derivatization of nonsulfonated tyrosines. Alternative MS/MS activation methods, including positive ion metastable atom-activated dissociation (MAD) and metal-assisted electron transfer dissociation (ETD) or electron capture dissociation (ECD) provide varying degrees of sulfonate retention. Sulfonate retention has also been reported following negative ion MAD and electron detachment dissociation (EDD), which also operates in negative ion mode in which sulfonate groups are less labile than in positive ion mode. However, an MS/MS activation technique that can effectively preserve sulfonate groups while providing extensive backbone fragmentation (translating to sequence information, including sulfonated sites) with little to no noninformative small molecule neutral loss has not previously been realized. Here, we report that negative ion CAD, EDD, and negative ETD (NETD) result in sulfonate retention mainly at higher charge states with varying degrees of fragmentation efficiency and sequence coverage. Similar to previous observations from CAD of sulfonated glycosaminoglycan anions, higher charge states translate to a higher probability of deprotonation at the sulfonate groups thus yielding charge-localized fragmentation without loss of the sulfonate groups. However, consequently, higher sulfonate retention comes at the price of lower sequence coverage in negative ion CAD. Fragmentation efficiency/sequence coverage averaged 19/6% and 33/20% in EDD and NETD, respectively, both of which are only applicable to multiply-charged anions. In contrast, the recently introduced negative ion ECD showed an average fragmentation efficiency of 69% and an average sequence coverage of 82% with complete sulfonate retention from singly- and doubly-deprotonated sulfopeptide anions.

Reduction in Database Search Space by Utilization of Amino Acid Composition Information from Electron Transfer Dissociation and Higher-Energy Collisional Dissociation Mass Spectra

With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.

How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ≈ 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified.

Carbonyl Charge Solvation Patterns May Relate to Fragmentation Classes in Collision-Activated Dissociation

Here, we investigate the hypothesis that the origin of Class I fragmentation in tryptic peptide dications corresponding to the cleavage of the first two amino acids from the N-terminus is due to a dominant charge solvation pattern. Molecular dynamics simulations (MDS) of model A(n)R dications confirmed the existence of a persistent solvation of the protonated N-terminus on the second backbone carbonyl. Additionally, MDS predicted a new distinct fragmentation class corresponding to the loss of two amino acids from the C-terminus. This prediction was confirmed experimentally at very low excitation levels. The pattern produced by electron transfer dissociation of the same dications gave markedly decreased cleavage frequencies at the second peptide bond, which, within the non-local fragmentation mechanism, supports the preferential charge solvation on the second carbonyl. Taken together, these results confirm the role of a charge solvation pattern in the origin of fragmentation classes.

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95% more (P=0.030) methylarginine-carrying peptides are selected for MS/MS than DDA, leading to an 86% increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6×10(4) cps) are selected for MS/MS using this approach relative to DDA (50% more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586% increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.

Comparison of CID, ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision-induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom-activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b-/y-type ions with abundant neutral losses of the phosphorylation modification. ETD produced c-/z-type ions in highest abundance but also showed numerous y-type ions at a frequency about 50% that of the z-type ions. The major peaks observed in the ETD spectra correspond to the charge-reduced product ions and small neutral losses from the charge-reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15-20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD.

ETD fragmentation features improve algorithm

Electron transfer dissociation (ETD) is an alternative technique used in mass spectrometry-based proteomics experiments. Because it is newer, most of the protein identification algorithms for ETD are still a simple derivation of well-established collision-activated dissociation algorithms without the consideration of many unique ETD spectral features. Sridhara and coworkers recently reported removing the charge-reduced precursors and corresponding neutral loss peaks to improve ETD peptide identification with the Open Mass Spectrometry Search Algorithm (OMSSA). These peaks were also used to deduce the charge of the precursors for low resolution data. The scheme is a concrete example of implementing known ETD fragmentation features to improve a protein identification algorithm.

Clinical Proteomics

Clinical proteomics involves the analysis of protein expression of disease proteomes, with the aim of solving a specific clinical problem. Discoveries made from proteomicbased studies contribute to the growing need for innovative medical diagnostics for disease detection. Taking into consideration the global health burden of cardiac disease, clinical proteomics is a valuable tool to improve risk stratification associated with this disease. In cardiovascular medicine, the identification of novel proteins, or biomarkers, that are differentially expressed in cardiac disease proteomes may enable early detection of the disease state, thereby preventing progression to disease end points. This review outlines various proteomic platforms and their technical advancements and relates these to the cardiovascular sciences. The entire protein complement of the cell, or proteome, is dynamic and changes in response to the disease state. 1 Proteomic-based experiments can be used to characterize such alterations in protein expression during disease progression. 2 With combined improvements in mass spectrometry (MS) technology as well as innovative molecular biology screening tools, there has been widespread growth in the characterization of cardiac disease proteomes. In fact, proteomic technology has been an important tool in the analysis of heart failure (HF), 3,4 cardiac hypertrophy, 5,6 and dilated cardiomyopathy.7 Several of the overarching aims of such studies include providing greater understanding of general biological mechanisms as well as identifying unique proteins that are clinically useful in the detection of cardiac disease in the early stages or potentially used as novel therapeutic targets. Clinical proteomics continues to benefit from advancements in technologies that allow for fast and consistent identification of proteins with corresponding increases in the dynamic range of proteins detectable in the disease proteome. MS-related technologies have improved in their ability to detect low-abundance proteins as well as membrane proteins and have benefited from sample preprocessing strategies that decrease the complexity of large-scale analyses. With the emergence of different methodologies for biomarker identification, the following 3 criteria must be taken into careful consideration to determine whether the protein is, in fact,