Advances in top-down proteomics for disease biomarker discovery

J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 9 2 0 – 9 3 4

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

Review

Advances in top-down proteomics for diseasebiomarker discovery

David Calligaris, Claude Villard, Daniel Lafitte⁎

INSERM UMR 911, Centre de Recherche en Oncologie biologique et en Oncopharmacologie, Aix-Marseille Université, Plateforme Protéomique etInnovation Technologique Timone, Faculté de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France

A R T I C L E I N F O

⁎ Corresponding author. Tel.: +33 49 183 5680E-mail address: [email protected]

1874-3919/$ – see front matter © 2011 Elsevidoi:10.1016/j.jprot.2011.03.030

A B S T R A C T

Article history:Received 19 January 2011Accepted 29 March 2011Available online 6 April 2011

Top-down mass spectrometry strategies allow identification and characterization ofproteins and protein networks by direct fragmentation. These analytical processesinvolve a panel of fragmentation mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry toprobe modified proteins as potential disease biomarkers. This review describes separatingmethods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects offragmentation mechanisms used for top-down analysis. The biological interest of thisstrategy is extensively reported regarding the characterization of post-translationalmodifications in biochemical pathways and the discovery of biomarkers. One has to bearin mind that quantitative aspects that are beyond the focus of this review are also of criticalimportant for biomarker discovery. The constant evolution of technologiesmakes top-downstrategies crucial players in clinical and basic proteomics.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Top-downFragmentationProteomicsBiomarkersPost-translational modifications

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9212. Top-down workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921

2.1. Separation and sample handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9212.2. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9232.3. Dedicated search engines for top-down analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 923

3. Fragmentation mechanisms for primary sequence analysis observed during top-down analysis . . . . . . . . . . . . 9253.1. Collision-induced dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9253.2. IRMPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9253.3. Electron capture dissociation and electron transfer dissociation . . . . . . . . . . . . . . . . . . . . . . . . . 9253.4. Laser-induced dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9283.5. MALDI in-source decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928

.(D. Lafitte).

er B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jprot.2011.03.030

mailto:[email protected]

http://dx.doi.org/10.1016/j.jprot.2011.03.030

921J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 9 2 0 – 9 3 4

4. Contribution of top-down approaches for characterization of biomarkers and biochemical pathways . . . . . . . . 9285. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930

Protein separation

Electrophoretic methods

2D gel electrophoresisContinuous-elution electrophoresis, GELFrEEMicrofluidic-based gel protein recovery system

Chromatographic methodsRPLC, HILIC, UHPLC

Ion exchange chromatographyChromatofocusing

Mass spectrometryMALDI-TOF

ISD

Q-TOF LTQ-Orbitrap FTICR

CID HCD IRMPDETD/ECD

Data analyses

ProSight BIG Mascot Spectra alignment approach

Fig. 1 – Overall view of top-down analysis.

1. Introduction

Proteomics approaches embeddingmass spectrometry (MS) arewidely used in biomarker research and discovery to findsignatures for disease diagnosis or prognosis [1,2]. Some ofthese biomarkers have been identified [3]. For example, Bon etal. showed that levels of decarboxyprothrombin and alpha-fetoprotein were increased in hepatocellular carcinoma andcould serve in diagnosing the disease [4]. Another study showedthat CYFRA 21-1 is a predictive factor of survival of non-smallcell lung cancer patients treated with gefitinib [5]. The mostwidely used ionization techniques for analyzing biomoleculesare electrospray ionization (ESI) and matrix-assisted laserdesorption ionization (MALDI) [for a review, see 6]. These softionization modes enable ionization of large biopolymers, suchas proteins, lipids, and nucleic acids. Two complementarystrategies based on ESI or MALDI ionization are used for proteinidentification. These approaches are named "bottom-up" [forreviews, see 7–12] and "top-down" [for reviews, see 13–19]mainly because of the initial digestion or not of the analyte. Inthe case of bottom-up approaches, proteins of interest undergoenzymatic digestion before MS analysis. This digestion can beperformed directly in solution, after separation by 1D- or 2D-gelelectrophoresis, but also by direct digestion on tissues. Peptidesobtained by enzymatic digestion, generally with a mass below3500 Da, are then directly analyzed by MALDI MS or ESI. Proteinidentity is then determined by the comparison, in this case, ofpeptide masses from MS analyses (peptide mass fingerprint byMALDI-TOF [for a review, see 20]) or MS/MS (tandem massspectrometry [for a review, see 21]), with peptide theoreticalmasses derived from proteomic or genomic databases. Thisapproach, the most widely used serves well for analyzingcomplex samples. However, separation by mono or bidimen-sional HPLC is needed to concentrate and separate peptidesissued from protein digestion. This strategy has two maindisadvantages. It is time-consumingbecauseproteinsmust firstbe digested, and it is limited because the protein sequence israrely exhaustively covered [9,22]. Moreover, ion signals for N-and C-termini fragments are not oftenmeasured, and fragmen-tation processes commonly used during bottom-up approachesinduce loss of labile post-translational modifications (PTMs)(phosphorylation, glycosylation…), which are potential bio-markers in clinical proteomics.

Top-down approaches seem more appropriate to directlyobtain, without preliminary digestion, protein N and C-termini.This strategy is often compared to Edman sequencing. Molec-ular ions from intact proteins are obtained by MALDI or ESIionization and fragmented by MS/MS. When ESI-MS is theionization method, Electron Capture Dissociation (ECD) andElectronTransferDissociation (ETD) [for a review, see 23] are themost suitable fragmentation techniques for top-down analysis.They are used for gas phase fragmentation involving electroncapture by an analyte to form a free radical with high energy-inducing peptides/proteins N–Cα bond cleavage and preserving

labile PTMs upon electron transfer. The fragmentation can alsobe achieved by collision of analyte molecules with an inert gassuch as heliumor argon. This process, which promotes a higherenergy pathway than ECD and ETD, is called Collision-InducedDissociation (CID) and often leads to the loss of labile PTMs [24].This dissociation can be achieved by another process such asphoto-fragmentation, which is induced by absorption of mul-tiple infrared photons (IRMPD for Infrared Multiphoton Disso-ciation). MALDI top-down is also possible by a relatively oldstrategy called In-Source Decay (ISD) updated for protein by theuseofMALDI-TOF/TOFmass spectrometers [for a review, see 25]with pseudo MS3 strategies.

In this review, we investigate the various top-down strate-gies available for protein biochemistry and their importance inthe clinical field (Fig. 1). We focus on the workflow, whichbasically includes instrumentation and bioinformatics, andtheoretical aspects of fragmentation processes (Table 1). Then,we examine the importance of top-down for the characteriza-tion of biomarkers and discuss how to improve top-downanalysis for in situ characterization of biopolymers.

2. Top-down workflow

2.1. Separation and sample handling

For top-down analysis, samples must often be made lesscomplex. Electrophoretic or chromatographic methods serveto separate proteins prior to top-down analysis [26,27] (Table 1).

Table 1 – Top-down proteomics workflow.

Proteins separation Electrophoretic methods 2D gel electrophoresis [30]Continuous-elution electrophoresis [30]Microfluidic-based gel protein recoverysystem

[31]

Gel-eluted liquid fraction entrapmentelectrophoresis (GELFrEE)

[40]

Chromatographic methods Reverse phase liquid chromatography(RPLC)

[30,32,33,35,104,106–108,110,111,114]

Hydrophilic interactionchromatographic (HILIC)

[34,37,111]

Ion exchange chromatography [36]Chromatofocusing [38,39]Ultra-high performance liquidchromatography (UHPLC)

[34]

Mass spectrometry Instruments ESI-triple quadrupole [42,43]Hybrid quadrupole TOF [44,45,106,114]LTQ-Orbitrap [33,48,61,62,104,106,119]FTICR [35,49,105,109]Hybrid quadrupole-FTICR [30,33,110,111]LTQ-FTICR [107,108]MALDI-TOF [50–54,87,89–105,112–120,124]

Fragmentation Collision-induced dissociation (CID) [63–75,105–107,119]Higher energy collisionally activateddissociation (HCD)

[61,62]

Infrared multiphoton dissociation (IRMPD) [76–78,105,109,111]Electron capture dissociation (ECD) [79–85,105,108,109,111],Electron transfer dissociation (ETD) [86,117]In-source decay (ISD) [50–54,87,89–100,112,114,120,124]

Data analyses Bioinformatics ProSight [56,57,104,107]BIG mascot [59]Spectra alignment approach [60]

922 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 9 2 0 – 9 3 4

In the case of mono or bidimensional electrophoresis, whichseparate proteins on the basis of their molecular weight andisoelectric point, the recovery of the proteins from gel spots isdifficult and often leads to protein fragmentation [28]. Thisrecovery performed via passive elution [29] or gel electroelutionis not readily compatible with ESI-MS because of the chemicalsused for protein extraction. Despite these technical limitations,Meng et al. characterized more than 400 proteins by massspectrometry after separation by 2D gel electrophoresis and off-line recovery into pools of 5–20 proteins. Proteins wereseparated in the first dimension by continuous-elution electro-phoresis, replacing SDS by an acid-labile surfactant (ALS "Acid-Labile Surfactant"), and then by Reverse Phase Liquid Chroma-tography (RPLC) in the second dimension. Characterization ofintact protein ions is done by on-line analysis with a 9.4Tquadrupole-Fourier Transform MS hybrid instrument [30]. Toovercome this problem, Hydrophilic Interaction Chromatogra-phy (HILIC) can also be performed after sample elution prior toMS. A new microfluidic-based gel protein recovery system,which uses a proprietary plastic microfluidic chip in a highvoltage environment, allows extraction from 10% “ALS-PAGE”gels of proteins with molecular weights between 6 and 65 kDaand is compatible with MALDI and ESI mass spectrometry [31].Chromatography is an alternative to electrophoreticmigrationsfor fractionation of intact proteins and top-down analysis. Thisseparation strategy can be done on-line with tandem massspectrometry using High Performance Liquid Chromatography(HPLC) or off-line with infusion of separated fractions of intactproteins. RPLC is themost often used stationary phase inmono

dimensional HPLC. Wang et al. explored parameters like alkylchain length, capillary temperature, or ion-pairing agent toenhance chromatographic resolution in intact protein separa-tion with reverse phase column [32]. They found that the bestconditions involved the use of C18 column heated to 60 °C andaddition of trifluoroacetic acid as ion-pairing agent. Theseoptimized parameters allow the same separation performanceof yeast cell lysates in their native or denatured form. Mono-dimensional separation does not provide enough peak capacityto resolve complex protein mixtures. However, Pesavento et al.used RPLC andmetabolic labeling coupled with high resolutionelectrospray mass spectrometry to separate protein isoforms,revealing progressive methylation of lysine20 in Histone-4during cell cycle [33]. A fast, automated, on-line methodinvolving Ultra-High Performance Liquid Chromatography(UHPLC) andmass spectrometrywas reported to profile histonemodifications and variants from human fibroblast [34]. Intactproteins separatedby reversephase capillary columncoupled toa Fourier Transform Ion Cyclotron Resonance (FTICR) massspectrometer revealed the presence of over 20 casein isoformsarising from genetic variants with different numbers ofphosphorylation sites [35]. To increase peak capacity and toresolve more complex mixtures of intact proteins, 2D chro-matographic strategies are coupled to high resolution massspectrometer detection. Reverse phase, highly compatible withelectrospray, is the second dimension of choice whether ionexchange [36], hydrophilic interaction chromatography (HILIC)[34,37], or chromatofocusing. [38,39] is used in first dimension.Another attractive and recent techniquecalledGELFrEE for "Gel-


Eluted Liquid Fraction Entrapment Electrophoresis" is used fortop-down analysis. The equipment includes a precision-castgel column surrounded by fraction collection chambers.Charged molecules are separated according to their molecu-lar weight. Proteins are then eluted into the fractionationcollection chamber where they are trapped in a defined liquidvolume. This technique allows rapid fractionation of intactproteins from 3.5 to 250 kDa [40]. Ionmobility devices are newseparating devices with the advantage of direct coupling tomass spectrometers [for a review, see 41]. This techniqueallows separation of ions in the gas phase not only by theirmass but also their shape. Isobaric proteins or peptides cantherefore be analyzed separately, which is not possible usingconventional mass spectrometry. Although there are noreports on coupling of ion mobility separation and top-down, this new technology could rapidly emerge as a powerfultool in the field.

2.2. Mass spectrometry

Early top-down analyses have been done on mass spectrom-eters equipped with quadrupole analyzers. Loo et al. studied a14-kDa protein such as ribonuclease A by using ESI-triplequadrupolemass spectrometers [42]. Feng et al. used the sametype of instrument for the analysis of larger proteins, such asan antibody (150 kDa) [43]. Nemeth-Cawley et al. used a hybridquadrupole TOF instrument to characterize a recombinant,immunoglobulin gamma-1 (IgG-1) fusion protein [44]. Al-though this study allowed isotopic mass assignment offragment ions with mass above 10 kDa, low resolution(between 8000 and 12,000) and accuracy (between 5 and20 ppm) limits the study of intact proteins to a mass of35 kDa. Instrument optimization is necessary to characterizeproteins of higher molecular weight. As an example, modifi-cation of the ion guide voltage (1–100 V) of an ESI-Q-TOFallowed the top-down study of N-terminal heavy (50-kDa) andlight (25-kDa) chains of a solution of gamma globulin [45].Beyond 35–40 kDa, however, Fourier Transform mass spec-trometers seem to be the solution [46,47]. These spectrome-

Fig. 2 – MALDI-reISD spectrum of HeLa cell tubulin. Fragment ionMALDI in-source decay.Monoisotopical specieswere assigned usy28-ion (m/z 3107.27 Da) and y28-ion (m/z 3253.24 Da) of α1B- an

ters, with resolution above 105 and accuracy below ppm levelare potentially the best instruments to obtain protein se-quences from top-down fragmentations patterns. Two typesof technologies encompass the term FT, orbital trap and ioncyclotron resonance. These mass spectrometers transformthe frequency of ion oscillation in a cell into molecularmasses. Orbitraps are used for analyses, by infusion, ofproteins with molecular mass not exceeding 50 kDa or, byRPLC-MS/MS, of proteins with molecular mass below 25 kDa[48]. Instruments such as FTICR can characterize proteins withnominal mass exceeding 100 kDa [49]. Several top-downstudies were also undertaken on MALDI mass spectrometers.Loo et al., for example, studied proteins from Escherichia colithrough a virtual 2 D strategy [50]. The proteins were firstseparated according to their Pi and then analyzed withoutelution or digestion directly in the gel by MALDI-TOF MS. Byusing this strategy, 2.5 times more proteins were detectedthan in classical 2D gel electrophoresis coupled to silverstaining. In addition, some proteins such as MetE protein, amethionine synthase of 84.6 kDa, were identified by MALDItop-down, also called MALDI in-source decay. Some studiesreported on MALDI in-source decay (ISD) for protein identifi-cation [51–54]. As an example, tubulin isotypes of HeLa cellswere characterized using ISD, which shows this technology isof interest for characterizing protein isoforms [54] (Fig. 2). Theemergence of MALDI-TOF–TOF mass spectrometers hasallowed ISD fragment sequencing by a pseudo MS3 techniquenamed T3-sequencing [51–54].

2.3. Dedicated search engines for top-down analysis

There are several search engines for identifying proteins forbottom-up approaches: Mascot™, X!tandem™, Sequest™,Phenyx™, and ProteinProspector™ are the most popular. Forpeptidemass fingerprint experiments, protein identification isdone by comparing experimental m/z values of enzymaticdigests with theoretical m/z values from in silico proteindigestion. Identification is also done by database searchingwith experimental MS/MS data sets of selected ions in the low

s corresponding to C-termini tubulin isotypes obtained bying BioTools 3.1 software. Insets show isotopic patterns of thed βI-tubulin isotypes, respectively.

B

CA

Fig. 3 – Mechanisms of fragmentation used in top-down proteomics. (A) Model of mobile proton explaining protonated peptidedissociation during CID fragmentation. Relocation of the N-terminus proton promotes the formation of an activatedintermediate, which leads to charge-directed amide bond cleavage. (B) Formation of cn- and zn-series ions via cleavage of atransient hypervalent radical produced by intermolecular hydrogen transfer under matrix-assisted laser desorption/ionizationconditions. (C) ETD fragmentation scheme of amultiply protonated peptide after reaction with a low energy electron to producecn- and zn-type ions.


mass range. These search engines are constantly improved todecrease the number of false positives (typical for “database-based” search engine) [55]. It has been proposed to combinesearches from various engines but such an approach has juststarted and is still at the stage of experimentation. Softwaresolutions for the analysis of intact protein fragmentationand top-down analysis are not largely developed. There isan increasing demand for software that allows web-basedidentification and characterization of proteins by directcomparison of mass list of observed parent and fragmentions against annotated proteomic databases. ProSight was thefirst web application for top-down identification of proteins[56,57]. This software corresponds to the combination ofsearch engines and a browser environment for the analysisof fragments above 10 kDa. ProSight is centered on organism-specific databases of protein- and site-specific informa-tion. Fragment ion masses of high mass parent ions are usedto performsearches against the constantly updated databases,which include post-translational modifications and “single

nucleotide polymorphisms” (SNPs). It is also possible todetect and localize not annotated PTMs by using a “Δmsearching mode” that considers an error tolerance (Δm)between protein experimental mass and potential candidates’theoretical masses. To take into account the possibility thatseveral PTMs, splice variants, or a variation in amino acidsequence due to polymorphisms of a single genome base pairor SNPs arepresent simultaneously, the softwarehas amethodthat is commonly referred to as “shotgun annotation” [58].Besides ProSight, new bioinformatic tools have been devel-oped. Mascot, the most used web-based database searchengine for bottom-up experiment due to its probability-basedscoring method and its fast operating algorithm, is limited to16 kDa for precursor ion selection. BIG Mascot has beendesigned for top-down analysis and is therefore used toidentify larger proteins fragmented by CID or ECD [59]. Anotherapproach is to align spectra in order to assign the differentisomers of a protein [60]. This algorithm can find optimalalignments between an experimental mass list issued from

image of Fig.�3


spectrum and a theoretical mass list of a protein sequenceby using a determined number of mass shifts that correspondto post-translational modifications, mutations, or insertions/deletions.

3. Fragmentation mechanisms for primarysequence analysis observed duringtop-down analysis

Fragmentation of biopolymers can occur inside or post themass spectrometer source through different mechanismscompatible with top-down analysis.

3.1. Collision-induced dissociation

The soft ionization sources currently used in biological massspectrometry generate ions in the gas phase by low energyprocesses. Collision of ionized molecules with an inert gas(helium, argon, nitrogen, or xenon) in the cell of the massspectrometer leads molecules to an excited state. Part of theions’ kinetic energy is converted into internal energy. Theseactivated ions undergo unimolecular decomposition thatleads to fragmentation. Fragmentation by CID can occur viahigh or low energy collisions. High-energy collisions can takeplace only in mass spectrometers with magnetic or TOF/TOFanalyzers where the energy involved is a few keV. In this case,ion fragmentation usually occurs in one step. Low energymass spectra are obtained by temporal or spatial tandemmassspectrometry, in which ion fragmentation occurs with acollision energy of a few eV. The collision cell is usually aquadrupole or a linear or 3D ion trap working in radiofrequency mode. Because ions’ residence time is longer andpressure in the collision cell is higher, fragments obtained bylow energy collision are the result of successive dissociations.Contrary to triple quadrupoles that provide a widemass rangefor fragment detection, ion traps suffers from lowmass cut off.Makarov and his group therefore upgraded the Curved LinearTrap (C-trap), a curved quadrupole where ions are stored byRF, coupled to the orbitrap with a higher energy collisionallyactivated dissociation (HCD) [61]. Top-down analysis of bovinecarbonic anhydrase II was obtained through fragmentation byHCD [62]. The CID process can also occur in the source; it iscalled in-source CID. Studies have shown that by varyingparameters such as voltage (source sampling cone, skimmer),temperature, pressure, drying gas, or at last the sourcegeometry, it is possible to fragment multiple charged ions ofhighmassmolecules [63–67]. This occurs because ions leavingthe capillary collide with the drying gas in the source. Thus,the increase in internal energy leads to ion fragmentation.This in-source fragmentation phenomenon is similar to thelow energy collisions obtained by tandem mass spectrometryin triple quadrupole analyzer. The advantage of this approachis the possibility to perform pseudo MS3 experiments frominstruments configured to perform tandem mass spectrome-try (MS/MS) [68,69].

The mechanism of CID fragmentation is usually explainedby the theory of the mobile proton [70] (Fig. 3A). In this model,it is the amino acids’ location and nature that influencepeptide or protein fragmentation. The addition of energy by

collisional activation leads to the relocation of the proton onexcitedmolecular ions. This relocation induces fragmentationat peptide backbones [70–74] (Fig. 3A). For most peptides orproteins, the fragmentation occurs at the amide bonds,leading preferentially to bn- and yn-series ions. High-energyCID induces additional fragmentation of residue side chains,generating vn- and wn-series ions. CID may favor certainschemes of fragmentation, such as amide bond rupture at N-terminal proline [71,74] and Gln–Gly bond cleavage [75].

3.2. IRMPD

During IRMPD dissociation, precursor ions undergo a series ofinfrared radiations by infrared photons, usually in the cell ofan FTICR mass spectrometer. This excitation enables ionheating and leads to the dissociation of peptide bonds. The ionfragments produced are of bn- and yn-series [76]. Thistechnique has the advantage over CID of preserving peptidephosphorylation [77]. IRMPD has also been used on otherinstruments. Raspopov et al. used IRMPD on a triple quadru-pole TOF to study top-down post-translational modificationsof a cancer biomarker, chaperonin-10 [78].

3.3. Electron capture dissociation and electron transferdissociation

In 1998 Zubarev et al. discovered ECD, a new process of peptideandprotein fragmentation [79]. Precursor ions are irradiatedwiththermal electrons (kinetic energy <0.2 eV) in the cell of FTICRmass spectrometers. Such fragmentation is exothermic (6 eV)and non-ergodic, which mean that the energy provided by therecombination necessary for fragmentation is not redistributedthroughout the molecule. Several devices produce the electronflow of electrons in ECD. The most widely used device iscomposed of a non-directly heated cathode irradiating for a fewms the precursor ions. Themain products of irradiation are [M+nH](n−1)+•. Another product of ECD is the [M+(n−1) H](n−1)+ ion.ECD induces the cleavage of parent ion N–Cα bonds, producing c′and z• daughter ions (Fig. 3B) or less frequently c• and z′ ions. Asecond fragmentation mode, leading to the formation of a• andy-type ions, can also be observed [79]. ECD fragmentation occursrandomly andprovides extensive coverage of peptides or proteinsequence [80]. However, there are some specific cleavagesinherent to this technique. Peptide cleavage at N-terminalproline is rarely observed [81] and disulfide bond disruption isfavored at the protein level [82]. Moreover, Fagerquist et al.showed that ECD is able to break disulfide bonds [83]. Electroncapture dissociation of peptides and proteins can also inducecleavage of certain amino acid side chains such as Lys, Arg, His,Asp/Glu, and Met [84]. Finally, ECD keeps PTMs (phosphoryla-tions, ubiquitination…) labile upon CID fragmentation [85].Although this phenomenon is poorly understood, it is probablydue to a lower intramolecular oscillation energy distribution,whichusually leads to the loss of thesemodificationsduringCID.

However, only 5 to 30% of precursor ions are converted intofragment ions and, because of the low kinetic energy ofelectrons for ECD, this approach is difficult to implement inmass spectrometers other than FTICR instruments [79]. So, anew technique emerged a few years ago to overcome theselimits while maintaining ECD's advantages. Electron transfer

Table 2 –Matrices for MALDI mass spectrometry.Matrix Structure Molecular mass (Da) λ (nm) Proton affinity (kJ/mol)

Alpha-cyano-4-hydroxycinnamic acid (CHCA)

HO CH C

CN

COOH

189.2 337, 355 8121, 8412

2,5-dihydroxybenzoic acid (DHB) HO COOH

OH

154.12 266, 337, 355 8523, 8554, 8562

3,5-dimethoxy-4-hydroxycinnamic acid (SA)

H3CO OCH3

OH

CH CH COOH 224.21 337, 355 8762, 9051

2,4,6-Trihydroxyacétophénone (THAP)

OH

HO

O

CH3

OH

168.15 266, 337, 355 8932

3-hydroxypicolinic acid (3-HPA)

N

OH

COOH

139.11 266, 337, 355 898.52

Trans-3-indol acrylic acid (IAA)

NH

COOH 187.2 337, 355 ND

926JO

UR

NA

LO

FPR

OT

EO

MIC

S74

(2011)

920–934

Dithranol (DIT) O OHOH 226.23 266, 337, 355 885.52

T-2-[3-(4-t-butyl-phényl)-2-méthyl-2-propénylidène]malononitrile (DCTB)

H3C

NC CN

t-Bu

250.34 266, 337, 355 ND

1,5-diaminonaphthalene (1,5-DAN) NH2

NH2

158.2 266, 337, 355 9425

5-aminosalicylic acid (5-ASA) NH2

OHO

HO

153.135 337, 355 8775

1, 2, 3, 4 and 5 Data from [120–124] respectively.

927JO

UR

NA

LO

FPR

OT

EO

MIC

S74

(2011)

920–934


dissociation developed by Hunt et al. in 2004 is mechanisticallyrelated toECDbutwasdesigned for instrumentswithan ion trapanalyzer, more widespread and less expensive [86]. Thistechnique is adaptable to mass spectrometers with 2D typelinear, spherical geometry or Orbitrap ion trap analyzers. ETDinvolves an electron transfer onto protonated proteins orpeptides (multicharged cations) from a reactive electron carrier(anion) formed outside the analyzer in a chemical ionizationsource. This source can be positioned opposite to the ESI sourceas in the case of LTQ-Orbitrap ETD or fitted between ESI sourceand the analyzer. Reactive species transfer to protonatedprotein/peptide occurs in gas phase by confinement of the twospecies in the analyzer. The fragmentation, which leads to thesame species as those in the ECDprocess, is a two-step reaction.First the electron transfer produces radical anion formation thatwill capture a proton. The result is the formation of an amino-ketal radical site that will trigger the dissociation of thecompound in cn- and zn-series ions (Fig. 3B).

3.4. Laser-induced dissociation

DuringMALDI ionization, ionized peptides/proteins can undergounimolecular reactions leading to the cleavage of the peptidebackbone. These dissociations are called metastable fragmenta-tions. Laser-Induced Dissociation (LID) corresponds to a frag-mentationof acceleratedmolecular ionsduring their travel in theflight tube (TOF) due to excess energy acquired in the sourceduring MALDI. The fragment ions, principally of yn-, an- and bn-series, have the same velocity as their precursor ion and reachtheselectiongateat thesametime. InMS/MSmode, ionswith thesame velocity are then selected according to their time of flight.Reflectron then allows the separation of the different ionsaccording to theirkinetic energy [87]. Reflectron,however, cannotrefocus in one step the ions with kinetic energy differenceshigher than 30%. For this, mass spectrometer manufacturershave designed TOF–TOF instruments equipped with “LIFT”technique [52]. Through an increase in kinetic energy of all ions(+19 keV), the LIFT cell reduces the kinetic energy differencebetween parent and fragment ions under the 30% threshold.Thus, a gridless two-stage reflectroncan focus, inonestep,wholeions accelerated after the LIFT cell. This technical progress is ofinterest because it decreases analysis time and enhances themass resolutionofparent and fragment ions. Inaddition, gridlesstwo-stage reflectron focuses the ion beam for better detection ofthe different ionic species, increasing sensitivity to the sub-femtomole level [52]. The curved-field reflectron can alsoperform MS/MS analysis in one step, but the absence ofreacceleration decreases mass resolution and sensitivity [52,88].

3.5. MALDI in-source decay

ISD fragmentation occurs in the hot MALDI plume afterion desorption/ionization steps. Indeed, the laser beam withfluence 5–20% above analyte ionization threshold leads tointermolecular transfer of hydrogen atoms between excitedmatrix molecules and carbonyl oxygen on the peptidebackbone [89–91]. The transfer depends on the hydrogenbonding network between matrix molecules and analytes.This fragmentation process preferentially gives cn- and (zn+2)-series ions [89] (Fig. 3C). Other ions of yn-, an-, xn-, and bn-series

can also be observed [90]. Numerous studies have beenperformed to determine the influence of the matrix and thematrix to analyte ratio in the ISD process. Thus matrices canbe ordered in function of their ability to donate protons. Themost widely used matrices for ISD have the followingclassification: picolinic acid (PA)>1,5 diaminonaphthalene(1,5-DAN)>5-aminosalicylic acid (5-ASA)>2,5-DHB>sinapinicacid (SA)>α-cyano-4-hydroxycinnamic acid (CHCA) [92,93]. Astudy has shown that the optimal matrix for inducing ISDfragmentation is a mixture of PA (20 mg/mL) and 1,5-DAN(saturated) with a ratio of 25/75 (v:v). Such a mix generatesalbumin (66 kDa) ion fragments ofmasses around 1000 Da [93].In addition, 1,5-DAN is a matrix with reducing properties,which makes it possible to study peptide/protein moleculeswith disulfide bridges [92,94,95]. This chemical, however, istoxic and can easily be replaced by 5-ASA [92]. These twomatrices produce mainly cn-, zn-, and yn-series ions, whereas2,5-DHB matrix, the most commonly used, and SA favor ionfragments of cn- and yn-series [96] (Table 2). In the case of 2,5-DHB, a study has indicated that the 5-hydroxy group plays animportant role in the ISD process [91]. Finally, the CHCAmatrix is the least able to induce ISD fragmentation andproduces yn-, bn-, and an-series ions [96]. This CID-likefragmentation is due to collisions between analytes in theearly step of ablation in the fragmentation process. Thisphenomenon is associated with analyte initial velocity duringdesorption. The lower the initial velocity, the higher thecollision rate and therefore the fragmentation. Matrix proper-ties take an important place in this process and a study hasproposed a classification of initial velocity of certain matrices:CHCA>THAP>SA>2,5-DHB [97]. To obtain proton transferbetween the matrix molecules and analytes, the matrix toanalyte ratio is set higher than in conventional MALDI analysis.Thus, Takayama et al. indicated that this ratio should bebetween 5000/1 and 10,000/1 [91]. Protein/peptide ISD fragmen-tation during top-down analysis can also be influenced byprimary and secondary structure elements. The gas phasebasicity (GB) of eachamino acid and therefore the proton affinity(PA) associatedwith it can influence biomolecule fragmentation[98]. Studies showed that the presence at polypeptide N- and C-termini of high gas basicity amino acids such as lysine, arginine,or glutamic acid is relatively important because they favorfragmentation [99,100]. Moreover, fragmentation depends onprotein sequence since in-source decay is almost impossible atthe N-terminus side of the proline due to its cyclic nature but isfavored when Glycine and Valine are present [91]. Finally, thesecondary structure, such as a-helix and β-sheet, inhibitsbackbone cleavage in ISD. Indeed, Takayama et al. showed thatMALDI-ISD of equine-apomyoglobin, a protein composed ofthree kinds of secondary structures, revealed that helix struc-tures can inhibit cleavage at amine bonds on the backbone [89].

4. Contribution of top-down approaches forcharacterization of biomarkers andbiochemical pathways

Top-down proteomic analysis is a major asset for discoveringbiomarkers because, by measuring the intact protein molecular

Fig. 4 – ISD fragmentation recordedonaporcine eye lens slice. Images correspond to ISD fragmentsat (A)m/z2003.4, (B)m/z2159.4,and (C)m/z2296.6ofβ-crystallinS, themajorprotein inporcineeye lens tissue. (D)Overlayof the threeprevious ion images. Imagesindicate that the distribution of the three ions recorded is highly similar in porcine eye lens. The black and the yellow circlesindicate the region where the mass spectrum has been extracted (high-intensity pixel). (E) ISD mass spectrum extracted from ahigh-intensity pixel. The tag and themasses indicated in red correspond to theN-terminuspart ofβ-crystalline S (c-ion series), thegreen ones to the C-terminus part (z-ion series). The red green-framed annotation indicates a peak for which the m/z ratiocorresponds to both the c- and z-ions. The ion peaks for which images are shown are labeled with letters A, B, and C.


weight, it preserves both the entire protein sequence and theintegrity of PTMs. Fagerquist et al. used a MALDI top-downapproach to identify biomarkers of several diseases, includinghemorrhagic colitis, hemolytic-uremic syndrome, and thrombot-ic thrombocytopenic purpura [101]. These biomarkers were E. coliproteins with masses around 10 kDa that could be submitted todirect MALDI MS/MS analysis. To allow protein identification,Fagerquist and collaborators used specific software with a novelmethod of peak assignment and a special scoring algorithm[101,102]. The same approach for result processingwas also usedby Demirev et al. for direct identification by MALDI-TOF/TOF MSof various Bacillus spore species. MS/MS identification of acidicsoluble proteins of low molecular weight, which can be specificbiomarkers of certain spores, allowed distinguishing Bacillusatrophaeus and Bacillus cereus [103]. Using the high resolutioninstrument LTQ-Orbitrap, Demirev et al. performed de novosequencing on intact proteins from bacteria and characterizednew protein sequences. It was then possible to quickly achieve aphyloproteomic classification of newly characterized microor-ganisms [104]. ESI-MS FTMS study by Ge et al. using CID, IRMPD,and ECD on proteins secreted by Mycobacterium tuberculosis

allowed the identification of 10 proteins (3 previously unknown)withmassesaround10 kDa [105]. Pfliegeret al. combinedbottom-up and top-down approaches in their proteomic study of thecollagen-like domain of a 25-kDa protein, the C1q subunit of thecomplement complex. They showed that it is possible tocharacterize different PTMs patterns of collagen-like domain ofC1q and unmodified residues implicated in structural assemblyof this macromolecular complex by LC–MS/MS on a hybrid triplequadrupole TOF mass spectrometer and by LC–MSn on an LTQ-Orbitrap, which also served for high resolution mass measure-ments of the intact protein and analysis of MSn mode fragmen-tation [106]. The top-down approachmay be useful for the studyof proteins with transmembrane domains, which are difficult tocharacterize by LC–MS/MS analyses of peptides obtained withconventional enzymatic protein digestion. Ryan et al. used FTMSto fragment by SORI-CID proteinswithmasses up to 35 kDa, suchas the voltage-dependent anion channel, bacteriorhodopsin andcytochromeb6f complex [107]. Zhang et al.used ECD to locate thephosphorylation sites of cardiac troponin I [108]. Ge et al. coupledECD with IRMPD dissociation to detect the four oxidation sitesresponsible for the inactivationof theenzymeprolyl-4-hydrolase,


which catalyses the hydroxylation of proline in procollagen [109].Contrary to the bottom-up approach, the top-down approachdetects K216 oxidation directly from a single spectrum. Thisinteresting work by top-down MS/MS focused on oxidationkinetics data of prolyl-4-hydrolase. In a top-down kinetics studyby FTMS, Zhai et al. reported on this-thiocarboxylate (this-SH), aprotein involved in the synthesis of the thiazole subunit ofthiamine phosphate. They pointed out that it was possible tocharacterize aminoacids involved in the formationof imineThiGfrom its substrate, deoxy-D-xylulose 5-phosphate [110]. Top-down analysis can be an asset to label-free quantify proteins.Pesavento et al. showed that ECD not only identifies acetylationsites, but also quantifies samples composed of more than 15isomers of histone H4 presenting one or more acetylations bymeasuring fragment ion abundance [111]. MALDI top-down is anefficient approach for protein isoform characterization. A recentstudycharacterized theβIII-tubulin isotype, apotential biomarkerof poor prognosis cancers [54]. This isotype was identified in asolution of HeLa cell tubulin by ISD sequencing of its C-terminus.ISD fragmentation is of interest in biomarker discovery since ithas made it possible to sequence the N- and C-termini ofubiquitin (8.6 kDa), myoglobin (17 kDa), and bovine serumalbumin (66 kDa) [93] and to analyze PTMs like glycosylation inpermethylated glycans prepared frommouse IgG1 [112]. Thus byupdating bioinformatics tools, ISD fragmentation appears apromising tool for direct protein profiling in cell lysate but alsofor thedirect studyof intact proteinson tissue slices as in thecaseof imaging MALDI mass spectrometry (IMSS). This techniquereveals the relative abundance of proteins and their localizationin tissue sections with high spatial resolution neighboring 50 μm[113]. A recent study reported that ISD fragmentation can beapplied to in situ identify proteins located by IMSS to avoidmaker delocalization due to tryptic digestion Althoughpromising in situ ISD is up to now limited to identification ofmajor proteins [114] (Fig. 4). Therefore microextraction andtandem mass spectrometry using bottom-up or top-downapproaches are still complementary to IMSS for proteinidentification. Using a combination of IMSS and RPLC-ESI-MS/MS, Lemaire et al. evidenced a potential ovary cancermarker corresponding to a fragment of the 11S proteasomeactivator complex named Reg-alpha [115]. Meistermann et al.used IMSS to localize biomarkers of toxicity induced bygentamicin in rat kidney sections [116]. The identification oftransthyretin, which accumulates in the renal cortex inresponse to treatment, was performed using tissue micro-extraction followed by RPLC-ESI-MS/MS analysis. Rauser et al.focused on the specific expression of proteins based onoverexpression of HER2, a membrane glycoprotein on thesurface of breast cancer cells [117]. ESI coupled to ETDfragmentation identified a cystein-rich protein 1, CRIP1, abiomarker that serves to accurately define HER2-positivefrom HER2-negative tissues by IMSS. Another study hasshown the relevance of a fragment of mitogen-activatedprotein kinase/extracellular signal-regulated kinase kinasekinase 2 in prostate cancer (PCa) from uninvolved prostatetissue (MEKK2) [118]. IMSS indicated that this fragment has ahigh expression profile in the PCa area and MALDI MS/MSanalysis allowed its identification. These studies showed thatmicroextraction can produce enough material for ms/msanalysis so that top-down can be envisioned. This was nicely

demonstrated by Lagarrigue et al. who combined MALDIimaging at 20 μMofmammalian testis and top-down analysisby CID MS/MS [119]. This allowed identification of a proteinmarker of development of germ cells within the seminiferousepithelium.

5. Conclusion

Top-down strategies are constantly being improved ininstrumentation, bioinformatics, and data processing. Theincreased sensitivity and resolution of MS instruments andthe adaptation of techniques now allow direct analysis ofproteins with molecular masses exceeding 100 kDa. Furtheroptimization of fragmentation methods such as ISD, ECD, orETD coupled to ESI and MALDI ionization now allowsextensive coverage of protein sequences, especially N- andC-termini. These regions are highly variablewithin a family ofproteins and are the sites of many labile PTMs. These PTMsare usually lost during conventional CID but not by ISD, EDC,and ETD. This feature combined with the possibility ofdirectly analyzing intact protein suggests that the top-downapproach is particularly suitable for locating and character-izing PTMs. However, to study complex mixtures for theidentification of disease biomarkers, several parameters needto be optimized, for example, the separation of proteins priorto mass spectrometry. The use of MALDI mass spectrometerscan be a solution to directly study tissue sections andconsequently locate and characterize biomarkers, but fist animprovement of in situ fragmentation capabilities andsecond a better data treatment are needed for such a complexanalysis.

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Advances in top-down proteomics for disease biomarker discovery