New proteomic developments to analyze protein isomerization and their biological significance in plants

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Review

New proteomic developments to analyze protein isomerization and their biological significance in plants

Philippe Grappin

^a,

⁎ , Boris Collet

^a

, Hongqian Yang

^b

, Denis Jallet

^a

, Laurent Ogé

^a

, Roman Zubarev

^b

aInstitut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Institut National de la Recherche Agronomique, F-78026 Versailles cedex, France

bKarolinska Institutet, Medicinal Biochemistry and Biophysics, SE-17 177 Stockholm, Sweden

A R T I C L E I N F O A B S T R A C T

Article history:

Received 17 January 2011 Accepted 28 April 2011 Available online 7 May 2011

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.

Keywords:

Isoaspartome Arabidospsis Protein L-isoaspartyl methyltransferase Aging

Electron capture dissociation

1. Introduction

Among posttranslational modifications, the spontaneous formation of isoaspartyl (isoAsp) residues in peptides is a major source of the conformational modifications occurringin vivo[1,2]. Susceptibility for IsoAsp formation in the peptide is influenced by protein structure[3]and environmental factors including pH, temperature [4] as well as external stressful constraints[5–7]. Mainly isoAsp formation (Fig. 1) arises from non-enzymatic asparaginyl (Asn) deamidation or aspartyl (Asp) dehydration[8,9]that occurs by nucleophilic attack of α-amino group of the C-terminal flanking amino acid on the Asn side chain carbonyl group or on the Asp side chain carboxyl group respectively[10,11]. The formedL-succinimide ring is then spontaneously hydrolyzed at either theα- or ß- carbonyl group (typical half life of 4 h at physiological pH) to engender a mixture ofL-isoAsp andL-Asp linkages in a ratio of 3:1 [8]. As a minor side-product, the racemized form of

L-succinimide could also generateD-isoAsp andD-Asp (Fig. 2).

Commonly, formation of atypical isoAsp sites is considered as unwanted damage that is frequently resolved by the degradation of the protein[12,13]. Alternatively, this fault in protein structure could be restored by isoAsp conversion into Asp form (Fig. 2) thanks to the enzymatic property of the proteinL-isoaspartyl-O-methyltransferase (PIMT) that methy- lesterifies the isoAsp side chain α-carboxyl group with the methyl donorS-adenosyl-L-methionine (AdoMet) cofactor that is transformed intoS-adenosyl-L-homocysteine (AdoHcy). The

newly-formed methylester group is then spontaneously hydrolyzed into succinimide intermediate[14,15], which is once again hydrolyzed to a mixture of Asp 15–30% and isoAsp 70– 85%. The iterative PIMT-catalyzed succinimide formation from isoAsp substrate gives rise to the replacement of isoAsp with Asp in the peptide[16].

Also, studies in animal systems strongly suggest that isoAsp formation in proteins as well as its conversion into Asp residue may act as molecular clock regulating cellular processes. A hypothesis that isoAsp formation could be involved in protein turnover was already proposed four decades ago[17–19]. Since then, recent results gave evidence that these posttranslational modifications contribute to apoptosis[20–23], chromatin remodeling[24], cellular matrix interaction in development [25], and gene regulation by controlling phosphorylation or myristoylation of signaling factors[26]. However the emerging view that protein isomerization is not only related to deleterious damage but could constitute a mechanism for useful modifications in protein structure in biological processes has to be more deeply investigated and is not at all documented in plants. From overall studies only 200 proteins subjected to isoAsp formation were identified. Most of them were discovered from protein isolation when the opportunity arose from human disease investigations or when their abundance and high sensitivity to isomerization have permitted their detection on electrophoresis gels. The paltry number of reports identifying isoAsp-containing proteins remains a hurdle to appreciate the importance of this metabolism in physiological mechanisms.

Characterization of isomerized proteins in a biological sample requires isomer separation, Asn/ and Asp/isoAsp relative quantifications, and isoAsp site localization in peptide sequences. The identification of Asn deamidation is possible with mass spectrometry (MS) since Asn and Asp (or isoAsp) and succinimide could be separated by a mass difference of 1 Da and 17 Da respectively. Nevertheless isoAsp and Asp residues have identical mass and very similar charge, and cannot be distinguished by these physicochemical properties. With the aim of extending the characterization of these unusual Asp isomers in peptides, analytical tools have been developed in most cases for mammal studies and are currently extended to plant systems with the objective of studying the biological involvement of isoAsp accumulation in proteins and of their control by PIMT.

PIMT reactive isoAsp-containing peptides could be quantified with a methylation assay using a commercially available bovine recombinant PIMT (Promega, Madison, WI) and HPLC analysis and have been successfully applied in Arabidopsis to demon- strate the biological implication of PIMT1 in limiting isoAsp accumulation in seed longevity[27,28]. Protein separation using 2-D electrophoresis and on-blot methylation labeling using recombinant PIMT to compare wild-type and PIMT deficient mutant mice allowed the first proteomic identification of isoAsp-containing proteins in brain tissues [29,30] and have been essayed to investigate Arabidopsis seeds [31]. Another interesting approach was to induce isomerization in proteins produced from an Arabidopsis cDNA library and to isolate altered proteins by the recombinant AtPIMT1 enzyme that is specific from one of the 2 PIMT genes encoded by the Arabidopsis genome[32]. In these methods isoAsp-containing proteins were isolated in vitro for their PIMT specificity in denaturing H₂O

NH₃

L-Succinimide L-Asn

L-Asp

L-isoAsp

15-30%

70-85%

Fig. 1–Mechanism of spontaneous L-isoaspartyl

formation. Deamidation of Asn and isomerization of Asp lead to the formation of an unstable succinimidyl ring that is spontaneously hydrolyzed to generate a mixture of Asp (15 to 30%) and of abnormal isoAsp (70 to 85%) residues at physiological pH.L-Asp,L-aspartyl;L-Asn,L-asparaginyl;

L-isoAsp,L-isoaspartyl.

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conditions and were subsequently characterized using MALDI- TOF and MS analysis. Nevertheless it is not known if PIMT targets all isoAsp-containing proteins in the proteome. Very recently, a development for protein fragmentation using Elec- tron Capture Dissociation (ECD) technology gives access to peptide isomer separation from crude protein extracts that could be analyzed simultaneously by Fourier transform mass spectrometry[33]. This novel approach has allowed analysis of the complete isoaspartome of human cells without any labeling by PIMT enzymatic assay. The first attempt to extend this advance with plants is very promising. These recent proteomic developments will permit new perspectives for investigating the consequences of external and intracellular factors on isomerization patterns at the proteome scale and for better understanding how the cell senses these modifications and uses them in controlling pathways of biological response. In this review we will compare these different proteomic approaches that will open a new avenue to describe mechanisms controlling isoAsp accumulation and their physiological implications in plants.

2. Investigating the control of isoAsp accumulation in plant tissues

In plants, PIMT activity has been localized with highest level in seed[34–36]. Because this resting organ of the plant has to survive a long period of time in soil waiting for dormancy

release and for suitable conditions reinitiating growth, the presence of PIMT has been proposed as a repair mechanism limiting age related damage in proteins. At the start, endogenous PIMT activity was measured by a vapor diffusion assay using a synthetic peptide containing isoaspartyl residues and radiolabeled S-adenosyl-L-[methyl-³H]methionine ([³H]AdoMet) as methyl source [15]. The quantification of PIMT activity, done by scintillation counting of [³H]methanol released from the methylated peptide, has a limited sensitivity and requires manipulating volatile radioactivity. Interests were mainly focused on PIMT activity or PIMT gene regulation during seed development and in response to abiotic stress [37,38], probably because methods to quantify isoAsp- containing proteins in extracts require the use of a recombinant PIMT enzyme. The only illustration of isoAsp accumulation has been reported by Mudgett et al.[38]for aged barley seeds using the human recombinant PIMT [39] with [¹⁴C]

AdoMet. Since, the commercial ISOQUANT detection kit (Promega, Madison,WI) has been developed for pharmaceuti- cal applications. The detection is based on a methylation assay using bovine recombinant PIMT and AdoMet cofactor.

Each PIMT-catalyzed isoAsp methylation converts the methyl donor AdoMet into AdoHcy byproduct that could be detected by reverse phase HPLC and quantify using the chromato- graphic peak area [28,40]. More recently, we have used this method to investigate isoAsp accumulation in pimt1 Arabi- dopsis mutant displayingPIMT1over-expression[27]. Access L-isoAsp

L-isoAsp-OMe

D-isoAsp D-Asp

D-Succinimide L-Succinimide L-Asp

CH3OH

AdoMet

AdoHcy

PIMT

15-30%

70-85%

Fig. 2–Mechanism of enzymatic repair by PIMT. Unstable succinimidyl ring is spontaneously hydrolyzed to generate a mixture of Asp (15–30%) and of abnormal isoAsp (70–85%) residues at physiological pH.L-Succinimide can also racemize to

D-succinimide that undergoes hydrolysis to generateD-isoAsp andD-Asp mixture (gray box). PIMT catalyzes the conversion of isoAsp into Asp (15–30%). Thus, several enzymatic cycles are needed to fully repair proteins. AdoMet,S-adenosyl-L-methionine;

AdoHcy,S-adenosyl-L-homocysteine;L-Asp,L-aspartyl;L-Asn,L-asparaginyl;L-isoAsp,L-isoaspartyl;L-isoAsp-OMe,

L-isoAspartyl-O-methylester.

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to isoAsp quantification was determinant to make the link between isoAsp accumulation and loss of seed vigor, and to establish the contribution of PIMT1 for seed survival in limiting isoAsp accumulation throughout seed maturation and germination (Fig. 3). Nevertheless, this approach does not permit the identification of isomerized proteins as well as PIMT substrates in complex protein samples and requires at least picomolar quantities of proteins.

3. Proteomic identification using 2-D

electrophoresis separation and on-blot methylation

First proteomic identification of proteins accumulating isoAsp were developed in 2006 using 2-D electrophoresis protein separation[29,30]with the aim of characterizingin vivoPIMT

targets in mouse brain extracts. Proteins that have been transferred from the 2-D electrophoresis gel to a PVDF membrane are labeled by on-blot methylation (OBM) using [³H]AdoMet and recombinant PIMT (Fig. 4). The autoradio- graphic template is used to localize the position of labeled proteins and to elute them from an equivalent 2-D PAGE replicate. Following tryptic digestion, isolated proteins are analyzed using MALDI-TOF MS or tandem MS. The use of PIMT knock-out mice accumulating higher level of isoAsp has allowed detection of abundant neuron specific proteins.

Regardless, this approach have shown good results as in mammals they identified targets previously found like synapsin [41,42], and tubulin [43,44]. This approach was recently applied to investigate the identification of isoAsp-containing proteins in Arabidopsis seeds [31]. The use of recombinant Arabidopsis PIMT1 enzyme for OBM did not give any signal while the use of recombinant human protein has allowed the detection and identification of 27 distinct proteins, most of them representing storage proteins known for their impres- sive abundance in seed. Similar results were obtained in our team using [¹⁴C]AdoMet to label PIMT substrates in Arabidop- sis seeds submitted to aging (Rajjou L, unpublished data). This approach provides a unique opportunity to detect labeled isoAsp-containing proteins in plants despite the low sensitivity of the method that could miss low abundant but important isoAsp-accumulating proteins. Also, it should be noted that in practice the 2-D PAGE approach merely allows the separation of proteins whose pI is restricted in the range of the used IPG strip (pH 4–8) and excludes from the analysis proteins whose molecular weight is under 20 kDa[30]. Moreover, the stressful conditions of high voltage and long time migration for protein separation are inducing sources ofin vitroisoAsp formation that have to be considered despite the use of NEPHGE by Zhu et al.[30]to reduce this experimental artifact. We note here that the denaturing SDS-PAGE gel in the second dimension exposes isoAsp that might be inaccessible for PIMTin vivo. To obtain a more comprehensive list of protein targets in Arabidopsis seed, the sensitivity of this approach has to be improved and a separate investigation of highly abundant storage proteins from soluble fraction is required in order to uncover minor proteins that were masked in the analysis. In these studies, sites of Asp isomerization were not localized in peptides.

4. Exploring low abundant proteins using phage display biopanning

As alternative to identify isoAsp formation in low abundant proteins from Arabidopsis seeds, Chen et al. [32] have developed a phage display biopanning method using a cDNA library from mature, dehydrated and germinated seeds. This technique avoids the step of protein extraction that could be a stressful condition increasing isoAsp accumulation in proteins [5–7]. The cDNAs ligated in expression vector were packaged into bacteriophages and stored 2 weeks at 4 °C to obtain isoAsp accumulation, and the produced Arabidopsis proteins (localized on the phage coat) were incubated with the Arabidopsis recombinant PIMT1 enzyme (rAtPIMT1), previously bound to a solid support. After several stringent washes, 0

1 2 3 4 5

Dry seeds WT pimt1-1

Relative mRNA level

0 20 40 60 80 100

0 3 6 9 12

Time of storage treatment at 40°C, 8% WC (weeks) Germination % 4 days after sowing

Time of storage treatment At 40°C, 8% WC (weeks) 0

200 400 600 800

0 6 12

L-isoaspartyl-containing acceptor proteins (pmol mg-1protein)

A

B C

Fig. 3 – Characterization of pimt1-1 mutant seeds upon storage treatment[27]. Wild-type (black) andpimt1-1(gray) dry mature seeds were submitted to a storage treatment performed for 12 weeks at 40 °C and 35% RH yielding a seed water content of 8%, similar to the water content of dry mature seeds.(A)Germination percentages from these seed samples were measured 4 days after sowing. Values are from four repetitions of 100 seeds (4 × 100) (mean±SD).(B)RT-PCR comparison ofPIMT1transcript accumulation in freshly harvested dry mature seeds. Means ± SD are shown (n= 3) (C)Quantitation ofL-isoaspartyl-containing

methyl-accepting substrates. Values are from three repetitions each using 100 mg of seeds (mean±SD).

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bacteriophages exhibiting potential PIMT target (still bound to rAtPIMT1) are used to infect bacteria that are plated and PCR- analyzed to recover Arabidopsis cDNA. This approach provided new putative PIMT1 substrates in seed context. This approach identified 17 targets including storage proteins, protective function as LEA (late embryogenesis abundant) or HSP (heat shock proteins), and proteins of translational machinery that have assigned functions that contribute to seed vigor and germination. Nevertheless, the isoAsp formation induced in the bacteriophage could differ compared to what happensin vivo when the seed is exposed to stressful environment. Also, only a small fragment of the native protein is translated and exposed on the phage coat with the impossibility to form secondary or higher order structures that may modulate isoAsp formation.

5. Accessing in vivo isoaspartome using MS technology

In the overall approaches described above isoAsp containing proteins have been identified as PIMT substrates, but it's far to be established that PIMT reactive proteins are representative of the isoAsp state of the proteome (isoaspartome). Recent developments in tandem mass spectrometry methods applied to electrospray-produced ions allow identifying peptides affected by isoAsp formation at the proteome scale from crude protein extract and to localize the isoAsp sites in the peptide sequence with a relative quantification of the modified peptides[33]. Even if Asn deamidation could be easily characterized with mass spectrometry since there is a mass difference between Asn and IsoAsp residues, distinguishing Asp and isoAsp residues by MS remains a challenge since both residues have identical masses.

Cournoyer et al.[45]have shown that peptide fragmentation by

Electron Capture Dissociation (ECD) produces isoAsp specific fragments that haven't been found for Asp peptides. Beside the conventional complementary fragmentsC_nandZ_l-ngenerated in the ECD N–C_αbond cleavage, theC_nU+58.0054 (C2H2O2) andZ_l-n

−56.9976 (C2HO2) fragments were found as specific signatures of the presence of isoAsp residues in peptides where n is the position of the isoAsp residue andlis the peptide length (Fig. 5).

Nevertheless false positive identification of the specific isoAsp signature with unrelated ionic species could not be excluded. To improve the reliability of isoAsp identification during high- throughput isoaspartome analysis, isoAsp-containing peptides are identified by high mass accuracy measurements of isoAsp- specific ECD fragments, and identifications are further validated by additional criterion such as adjacent c/z fragments, chro- matographic peak shape, CO2loss from adjacent c/z fragments, presence of the complementary isoAsp specific fragment, or specific loss of Asp from reduced species. These parameters have been incorporated into software to automate the detection of isoAsp fragments (Fig. 6). This development of the method by Yang et al. [33] has permitted the validation of 219 isoAsp candidates among 419 that were provided by 32 proteomic datasets obtained from ECD analyses of human cell protein extracts. We have applied this approach to compare Arabidopsis aged seeds and a freshly harvested sample (Zubarev R and Grappin P, unpublished data) and preliminary results exhibit that ECD MS/MS applied to electrospray ionization is reliable for proteomic identification and quantification of isoAsp-containing peptides in plants. Among 619 identified proteins 22 isoaspartyl-containing peptides were identified and 16 peptides were differentially isomerized between fresh and aged seed samples. Interestingly one of the characterized isoAsp-containing peptides was previously identified using the phage display approach [32]. However, a significant part of the peptides targeted by isoAsp accumulation in aged seed sample are Protein Extraction

On-Blot Methylation (OBM) and Fluorography

Spots Excision

and MALDI-TOF Mass Spectrometry

Isoelectric Focusing (IEF)

2-D SDS-PAGE Separation

Coomassie or Silver Staining Transfer to PVDF Membrane

Fig. 4–Identification of rPIMT substrates using on-blot methylation approach. Following charge and mass separations, proteins are either stained in gel or transferred to PVDF membrane to undergo on-blot methylation. After substrate identifications, spots are excised from strained gels and characterized by MALDI-TOF mass spectrometry analysis.

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represented by HSP or LEA proteins, and by assigned function related to redox homeostasis of the cell. In this analysis, seed storage proteins represent 83% of the total protein abundance.

For further increasing the number of proteins analyzed we intend to reduce the amount of storage proteins in extracts using fractionation methods. In this analysis, Arabidopsis seed extraction methods have been adapted with protocols used for mammals to perform protein denaturation and tryptic digestion in the same buffer (RapiGest, Waters, Milford, MA) that is also compatible for reverse phase HPLC separation. The characterized isoaspartome allows identifyingin vivoisoAsp formation that occurs in the physiological context of the seed. Neverthe- less this approach doesn't avoid experimental artifacts inducing additional deamidations during protein extraction and tryptic

digestion. The use of H218O labeling[46]offers the possibility to discriminate the isoAsp accumulated throughout the protein extraction and the mass spectrometric analysis, and is promising to selectively identify the isoaspartome fromin vivoisoAsp formation. These developments suggest the ECD MS/MS approach will provide a very reliable and sensitive analytical tool for high throughput analysis ofin vivoisoAsp formation at the proteome scale.

6. Conclusion

The difficulties in separating and characterizing proteins modified by isoAsp formation have limited the exploration of [32].

Fig. 5–Example of a typical Spectrum with an isoAsp specific ECD fragment. ECD MS/MS spectra for LIIisoDSLYK. *D, theZ^#₅pic indicates the presence of isoAsp by the unique fragmentZ₅-57[33].

Fig. 6–High throughput isoaspartome analysis using Electron Capture dissociation combined with Fourier transform MS. The isoaspartyl peptides can be distinguished based on specific detection of the accurate fragment masses. The developed workflow allows for further enhancement of the detection rate of isoaspartyls in biological samples[33].

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the biological significance of these conformational changes in plants. The importance of PIMT in modulating these conformational modifications should make this pathway very reactive for plant response to environmental changes. Interestingly a recent genetic study in Arabidopsis discovered that the enzymatic control of protein isomerization by the PIMT enzyme is a key controlling mechanism of stress response[27]. Recent efforts to develop large-scale analysis using proteomic approaches pave the way to better understanding the biological importance of the isoaspartome. Particularly, recent mass spectrometry developments using ECD MS/MS provide a reliable method for sensitive and quantitative identification of isomerized residues in peptides from a complex protein mixture. The combined use of these novel proteomic approaches with PIMT deficient mutants in Arabidopsis is promising to decipher the regulatory pathways of protein isomerization controlling plant adaptation to stressful environmental factors.

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New proteomic developments to analyze protein isomerization and their biological significance in plants

Review