Article No. jmbi.1999.3415 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 295, 853±864

Post-translational Modification of p53 Protein inResponse to Ionizing Radiation Analyzed byMass Spectrometry

Jacinth Abraham1, John Kelly2, Pierre Thibault2 and Sam Benchimol1*

1Ontario Cancer Institute/Princess Margaret Hospital andDepartment of MedicalBiophysics, University ofToronto, 610 UniversityAvenue, Toronto, OntarioCanada M5G 2M92Institute for BiologicalSciences, National ResearchCouncil, 100 Sussex DriveOttawa, Ontario, CanadaK1A 0R6

E-mail address of the correspondbenchimo@oci.utoronto.ca

Abbreviations used: IR, ionizingtriethylamine; DNA-PK, DNA-depeMS, mass spectrometry; MALDI-TOlaser desorption ionization time ofspectroscopy.

0022-2836/00/040853±12 $35.00/0

The p53 tumor suppressor protein promotes cell cycle arrest or apoptosisin response to DNA damage and other forms of stress. p53 protein func-tions as a transcription factor by binding to speci®c DNA sequences andregulating the transcription of target genes. This activity of p53 isreported to be regulated by phosphorylation and acetylation occuringat various sites on the molecule. Here, we have used a direct andnon-radioactive approach involving mass spectrometric analysis of p53protein to identify sites that are covalently modi®ed in vivo, either consti-tutively or in response to ionizing radiation. Following partial puri®-cation by immuno-af®nity chromatography and enzymatic in-geldigestion, the resulting p53 peptides were analyzed by MALDI-TOF andnanoelectrospray mass spectrometry. Mass spectrometry analyses ident-i®ed four sites at the N terminus that were phosphorylated in responseto irradiation, a single constitutive phosphorylation site at serine 315 andseveral acetylation sites.

# 2000 Academic Press

Keywords: p53; phosphorylation; acetylation; mass spectrometry; post-translational

*Corresponding author

Introduction

The p53 tumor suppressor protein plays a criticalrole in the cellular response to DNA damage lead-ing to cell cycle arrest or apoptosis depending oncell type and culture conditions. Loss of the p53-dependent DNA damage response could lead togenomic instability and the survival of cells carry-ing mutations and carcinogenic lesions therebycontributing to malignancy (Levine, 1997). DNAstrand breaks produced by ionizing radiation (IR)or by DNA repair intermediates following treat-ment with UV-radiation or chemotherapeuticagents result in the accumulation of p53 proteinand in the activation of its transcriptional activity(Fritsche et al., 1993; Lu & Lane, 1993; Hupp et al.,1995). While elevated levels of p53 protein arebelieved to be important to initiate the events thatlead to G1 arrest or apoptosis after DNA damage,

ing author:

radiation; TEA,ndent protein kinase;MFS, matrix-assisted

¯ight mass

there is compelling evidence that post-translationalmodi®cation of p53 protein is also required to acti-vate the latent sequence-speci®c DNA binding andtransactivation functions of p53 (reviewed byGiaccia & Kastan, 1998; Prives, 1998).

Whether p53 protein is a direct sensor of DNAdamage involved in the initiation of the DNAdamage response pathway or a more distal partici-pant in the pathway is not known. One model pro-poses that p53 protein is activated post-translationally by surveillance proteins capable ofsensing DNA damage (Anderson, 1994). Attractivecandidates that may regulate p53 activity includeDNA-dependent protein kinase (DNA-PK), ATMprotein, ATR protein, stress activated proteinkinase (SAPK/JNK) and poly(ADP-ribose) poly-merase. All of these proteins are known to post-translationally modify p53 protein in vitro.

Previous studies using two-dimensional gel elec-trophoresis showed that p53 protein exists as sev-eral isoforms that differ with respect to charge invarious cell types. Using this procedure, we pre-viously reported that novel p53 isoforms appear incells that have been exposed to IR. The phospha-tase sensitivity of the radiation-induced p53 iso-forms in mouse kidney cells indicated that these

# 2000 Academic Press

854 Mass Spectrometric Analysis of p53 Modi®cations

different isoforms arose as the result of multiplephosphorylation events (Abraham et al., 1999).

p53 is phosphorylated by several kinases in vitro.Among these are Casein kinase I-like enzyme(mouse only, human equivalents Ser6, Ser9) (Milneet al., 1992; Knippschild et al., 1997), DNA-PK(Ser15, Ser37) (Lees-Miller et al., 1992; Shieh et al.,1997), ATM (Ser15) (Canman et al., 1998; Baninet al., 1998), ATR (Ser15, Ser37) (Tibbetts et al.,1999), CAK (Ser33 and undetermined sites at the Cterminus) (Ko et al., 1997; Lu et al., 1997), raf (sev-eral sites within ®rst 27 amino acids) (Jamal & Ziff,1995), SAPK (mouse only, human equivalentSer37) (Hu et al., 1997; Adler et al., 1997), MAPK(mouse only, Thr73, Thr83) (Milne et al., 1994),PKA (between amino acids 97 to 155) (Adler et al.,1997), PKC (Ser378) (Takenaka et al., 1995; Baudieret al., 1992), cyclinB/cdc2 and cyclinA/cdk2(Ser315) (Bischoff et al., 1990; Wang & Prives,1995), Casein kinase II (Ser392) (Meek et al., 1990;Adler et al., 1997), p38 kinase (mouse only, humanequivalent Ser392) (Huang et al., 1999) anddsRNA-PKR (Ser392) (Cuddihy et al., 1999). Theuse of phospho-speci®c antibodies has identi®edseveral serine residues on p53 that are phosphory-lated in vivo: Ser15 (Shieh et al., 1997), Ser20 (Shiehet al., 1999; Unger et al., 1999a), Ser33 (Ko et al.,1997), Ser37 (Sakaguchi et al., 1998), Ser376(Waterman et al., 1998), Ser378 (Waterman et al.,1998) and Ser392 (Lu et al., 1998). Phosphorylationat Ser315 was determined using radiolabelling andphosphopeptide/phosphoamino acid analysis.

p53 is acetylated in vitro by PCAF (Lys320) (Liuet al., 1999; Sakaguchi et al., 1998), and by p300(Lys373, Lys382) (Sakaguchi et al., 1998; Liu et al.,1999; Gu & Roeder, 1997). The in vivo acetylationof these sites was determined by acetyl-speci®cantibodies raised against those sites. The kinasesand acetylases that modify p53 protein in vivoremain unknown. Phosphorylation at the N termi-nus is implicated in regulating p53 protein stability(Shieh et al., 1997; Unger et al., 1999a). Phosphoryl-ation and acetylation at the C terminus areinvolved in modulating the DNA-binding functionof p53 (Hupp et al., 1992; Lu et al., 1997; Wang &Prives, 1995; Waterman et al., 1998; Sakaguchi et al.,1998; Gu & Roeder, 1997; Liu et al., 1999).

Here we have used mass spectrometry to charac-terize post-translational modi®cations of the p53protein in human cells before and after exposure toIR. The acute myeloid leukemia cell line, OCI/AML-3, was used as a source of p53 protein, sincethese cells were shown previously to express wild-type p53. The p53 protein in these cells hassequence-speci®c DNA binding activity and thisactivity increases following IR (Sutcliffe et al.,1998). Moreover, the p53-responsive gene,p21WAF1, is transcriptionally activated followingirradiation and the cells arrest in the G1 phase ofthe cell cycle (Fu & Benchimol, 1997). All of these®ndings indicate that the p53-responsive DNAdamage pathway is intact in OCI/AML-3 cells.Here, we show that phosphorylation at Ser315 is

constitutive and is not changed in response to IR.This study also provides independent con®rmationof previous phosphorylation and acetylationassignments on p53 and identi®es an additionalputative site for acetylation.

Results

Different isoforms of p53 in control andirradiated OCI/AML-3 cells

The different isoforms of p53 protein are easilydistinguished by 2D gel electrophoresis (Abrahamet al., 1999). p53 proteins with altered mobility canalso be observed in standard polyacrylamide/Tris-glycine gels using lower concentrations of acryl-amide and extended separation (data not shown).Electrophoresis on a denaturing 10 % polyacryl-amide/Tris-tricine gel provides better resolutionand revealed the presence of p53 protein withdecreased mobility in irradiated OCI/AML-3 cells(Figure 1(a)). These cells were shown previously toexpress wild-type p53 mRNA on the basis ofsequence analysis of the entire p53 coding region(Fu et al., 1996). The different isoforms of p53 weremore readily apparent when p53 was resolved onan 8 % polyacrylamide/Tris-tricine gel, extractedfrom consecutive gel slices and visualized byWestern blotting on a standard, denaturing 9.5 %polyacrylamide/Tris-glycine gel (Figure 1(b)).Although p53 isoforms, differing with respect tomobility, were present in both the control and irra-diated cells, the relative proportion of these iso-forms appeared to change after irradiation. Theseresults indicate a change in the p53 protein mass/charge ratio following IR, pointing to post-transla-tional modi®cations.

Preparation of p53 protein samples foranalysis by mass spectrometry

To identify the post-translational modi®cationsof p53 protein that might be giving rise to the var-ious p53 isoforms in both control and irradiatedOCI/AML-3 cells, we prepared samples suitablefor analysis by mass spectrometry. Extracts wereprepared from 60 � 108 non-irradiated cells and15 � 108 irradiated cells. We expected to retrievesimilar amounts of p53 protein from these cellssince previous experiments indicated a fourfoldincrease in the level of p53 protein, two hours afterexposure to 6 Gy radiation.

p53 protein was partially puri®ed by af®nitychromatography using Sepharose beads cross-linked to the p53 speci®c monoclonal antibodyPAb1801, as described in Materials and Methods.After extensive washing under stringent conditions(0.5 M NaCl), p53 was eluted with 50 mM triethyl-amine (TEA), pH 11.5. High base elution gave bet-ter yields of p53 protein from the af®nity columncompared with acid elution or antibody-epitopespeci®c peptide elution. The TEA eluate containedmany contaminating proteins amongst which

Figure 1. Multiple forms of p53 protein from OCI/AML-3 cells as identi®ed by one-dimensional SDS-PAGE.(a) Western analysis of p53 protein immunopurifed from control (CON) and irradiated (IR) OCI/AML-3 cells separ-ated on a 10 % Tris-tricine SDS-polyacrylamide gel. (b) Western analysis of p53 protein, extracted from consecutiveslices of a 8 % Tris-tricine SDS-polyacrylamide gel, showing the different isoforms present in control (CON) and irra-diated (IR) OCI/AML-3 cells. Extraction of p53 from the gel slices involved mincing the gel slices, soaking them in1 � SDS buffer and heating at 100 �C for ®ve minutes. Forms of p53 protein, other than the primary p53 bandextracted, possibly represent degradation or other such alterations incurred during the extraction procedure.

Mass Spectrometric Analysis of p53 Modi®cations 855

p53 represented a minor constituent (Figure 2(a)).Proteins in the TEA fraction were resolved by elec-trophoresis on a denaturing 5 % to 20 % polyacryl-amide gradient gel, which served to collapse thep53 isoforms into one predominant band. The con-taminating proteins did not pose any problem forsubsequent analysis, since they were removed bySDS-PAGE and gel puri®cation of p53. An aliquotof the sample was used to estimate the amount ofp53 protein relative to a known amount of seriallydiluted recombinant p53 protein (Figure 2(b)). Weroutinely recovered 200-500 ng of p53 by thisimmuno af®nity puri®cation procedure. Individualpreparations of p53 served as the starting materialfor subsequent in-gel tryptic digestion and massspectrometry analyses.

MALDI-TOF mass spectrometry

The tryptic peptides were extracted from the gelpieces and analyzed by MALDI-TOFMS. Figure 3shows the amino acid sequence of p53 and thesequence coverage of unmodi®ed p53 proteinobtained by tryptic mapping using MALDI-TOFMS. The tryptic map was similar for bothrecombinant and immunopuri®ed p53 and coveredabout 62 % of recombinant p53 and 69 % of OCI/AML-3 p53 protein. A number of peptidesobtained from the immunopuri®ed OCI/AML-3sample could not be assigned to p53. A search ofthe SWISSPROT/TrEMBL protein database(ExPASy) using the web-based algorithm, PeptideSearch (EMBL, Heidelberg), identi®ed b-tubulin as

the principal contaminant of these samples. Thiswas con®rmed by MS/MS analysis. By comparisonof the observed masses and the predicted massesof p53 tryptic fragments, it was possible to identifyp53-derived peptides that were likely to be post-translationally modi®ed. A summary of theMALDI-TOF mass spectral data is shown inTable 1.

The upper mass regions of the spectra, represent-ing peptides above m/z 2000, are shown inFigure 4. A small peak was observed at m/z 2819.4in the control, and at m/z 2819.1 in the irradiatedsamples corresponding in mass to the mono-acetyl-ated version of the N-terminal p53 tryptic peptide(T1-24). This likely represents acetylation of the ®rstmethionine, since many proteins bear this type ofmodi®cation (Bradshaw et al., 1998). A larger peakat m/z 2899.1 and a smaller one at m/z 2979.1were observed in the irradiated sample only andare likely to be mono- and di-phosphorylated ver-sions of this peptide, differing by the addition of80 and 160 mass units, respectively. Two serineresidues in this region of human p53, Ser15 andSer20, are known to be phosphorylated followingDNA damage by IR (Siliciano et al., 1997; Shiehet al., 1999; Unger et al., 1999a). These reports areconsistent with our results. Further con®rmation ofphosphorylation at this acetylated peptide, T1-24,was obtained by nanospray mass spectrometricanalysis and is presented in the next section.

A peak in the spectra was observed at m/z4596.2 in the control and irradiated samples corre-

Figure 2. Partial puri®cation of p53 protein from OCI/AML-3 cells using immunoaf®nity chromatography.(a) Lysates from control (CON) and irradiated (IR) OCI/AML-3 cells were mixed with Sepharose beads cross-linkedto the p53 antibody, PAb1801. The eluate from the PAb1801 af®nity column was subjected to electrophoresis on a5 %-20 % gradient SDS-polyacrylamide gel in preparation for in-gel tryptic digestion. A non-®xing silver stainingmethod was used to visualize the protein bands (Shevchenko et al., 1996). An arrow indicates the location of p53 pro-tein. (b) Western analysis to estimate the quantity of p53 protein used in in-gel tryptic digests (a). A small aliquout ofthe eluate from the PAb1801 af®nity column was separated on a 5 %-20 % gradient SDS-polyacrylamide gel alongwith a serial dilution of recombinant p53 protein ranging from 0.1 to 50 ng. Note the slower mobility of theHis-tagged recombinant p53 protein. The bands migrating below p53 are likely degradation products of p53 protein.

856 Mass Spectrometric Analysis of p53 Modi®cations

sponding to the p53 tryptic peptide T25-65. How-ever, only the irradiated sample exhibited a peakat m/z 4676.5, corresponding to the singly phos-phorylated version of this peptide. Furthermore,analysis in negative ion mode also revealed a smallpeak corresponding to the doubly phosphorylatedversion of this peptide (data not shown). Althoughthere are three serine and one threonine residues inthis peptide that could serve as acceptor sites forphosphate, the mass spectral data are consistentwith previous reports showing that Ser33 andSer37 become phosphorylated in response to radi-ation-induced DNA damage.

A peak at m/z 1666.5 (data not shown) maycorrespond to the tryptic peptide T306-320 with anacetyl group (Table 1). Trypsin will not cleave afteran acetylated lysine residue (Rice et al., 1977), andtherefore, we suggest that Lys319 is acetylated.Lys320 was previously shown to be acetylatedin vivo (Liu et al., 1999; Sakaguchi et al., 1998); inaddition, Lys319 and Lys321 may also be modi®edto a lesser extent (Liu et al., 1999). Two peaks atm/z 973.9 and 1199.1 may represent peptidesT380-386 and T373-382 that have been acetylated. Aprevious publication (Gu & Roeder, 1997) reportedincorporation of radiolabelled acetate at Lys370,

Lys372, Lys373, Lys381 and Lys382, with Lys373and Lys382 being the predominant acceptor sites.While MALDI-TOFMS results are usually substan-tiated by other mass spectrometry analyses, thepostulated acetylations presented in Table 1 do nothave additional con®rmations, and therefore, mustbe considered as tentative. The inferred acetylationof peptides spanning amino acids 373 to 386 wereonly observed in the irradiated samples. Our datalend support to a model by Gu & Roeder (1997), inwhich the positive charge of the C terminus of p53protein inhibits the ability of p53 to bind DNA andtransactivate target genes. They proposed thatacetylation at the C terminus of p53 protein servesto overcome this inhibitory effect by neutralizingthe positive charges.

Phosphopeptide and MS/MS analysis

The in-gel digest extracts were desalted asdescribed in Materials and Methods, and phospho-peptides were identi®ed by precursor ion scanningusing a triple quadrupole mass spectrometerequipped with a nanoelectrospray ionizationsource. A strong phosphopeptide signal wasobserved at m/z 965 in the precursor ion spectrum

Figure 3. Schematic representation of amino acid coverage of p53 protein obtained by MALDI-TOFMS. The pep-tides of recombinant p53 (broken lines above the amino acid sequence) and unmodi®ed OCI/AML-3 p53 (linesbelow the amino acid sequence) identi®ed by MALDI-TOFMS are shown. The peptide spanning the ®rst 24 aminoacids (*) is shown here though it was acetylated. Recombinant p53 had an additional 24 amino acids at its N termi-nus which included the His-tag, and was not seen. Lines that overhang at the margins denote continuity, represent-ing peptides that span a stretch of protein sequence that is broken into two lines in the schematic. Trypsin cleavagesites are shown in bold.

Mass Spectrometric Analysis of p53 Modi®cations 857

of p53 present in the irradiated sample(Figure 5(d)). This triply deprotonated ion corre-sponds to the singly phosphorylated and acetyl-ated T1-24. The small signal observed at m/z 1448.5is the doubly deprotonated version of this samepeptide. Unfortunately, MS/MS analysis failed toproduce an interpretable spectrum for this phos-phopeptide. Nevertheless, this result con®rms thatthe peak observed at m/z 2899.1 in the MALDI-TOFMS spectrum of this digest is a phosphopep-tide (Figure 4). The doubly phosphorylated versionof this peptide was not observed by precursor ion

scanning, most probably due to losses incurred atthe desalting step.

A second phosphopeptide was observed at m/z710 in the control and irradiated OCI/AML-3sample digests (Figure 5(c) and (d)). Thisdoubly charged ion corresponds to T307-319

(ALPNNTSSSPQPK) plus a single phosphategroup. MS/MS sequence analysis of this ion(Figure 6) supported this proposal. The exactlocation of the site of phosphorylation, however,could not be identi®ed. This result is consistentwith radiolabel-aided identi®cation of Ser315 as a

Table 1. Assignment of the peptide masses observed in the MALDI-TOFMS spectra of the in-gel tryptic digestextracts of control and irradiated p53 protein

Observed mass (OB)

PeptidePredicted

mass (PR)a Con Rad �Mb (PR-OB) Comments

T197-202 687.4 - 687.1 0.3T159-164 696.4 697.2 - 0.8T338-342 729.3 729.5 729.5 0.2T268-273 751.4 751.6 751.5 0.2T66-72 771.4 771.7 771.5 0.3T274-280 819.4 - 818.5 0.9T133-139 897.4 897.6 897.6 0.2T380-386 931.6 - 973.9 42.3 Possible acetylation; previous report at K382T336-342 1014.5 - 1014.8 0.8T111-120 1030.6 1030.8 1030.7 0.2T283-290 1046.5 1046.9 - 0.4T243-251 1058.6 1059.0 - 0.4T102-110 1078.5 1078.7 1078.6 0.2T373-382 1156.6 - 1199.1 42.5 Possible acetylation; previous report at K373T165-174 1214.6 1215.1 1215.0 0.5T283-292 1302.7 1302.2 - 0.5T307-319 1340.7 1341.2 - 0.5T293-305 1385.7 - 1386.2 0.5T203-213 1427.7 1428.3 1428.1 0.6T268-280 1551.7 - 1552.4 0.7T306-320 1624.9 1625.5 - 0.1T306-320 1624.9 1666.5 - 41.6 Possible acetylation at K319T291-305 1641.9 1642.4 1642.4 0.5T320-333 1707.9 1708.2 1708.1 0.3T291-306 1798.0 1798.3 1798.1 0.3T140-156 1910.9 1911.3 1911.1 0.4T249-267 2068.2 2068.5 2068.3 0.3T197-213 2096.1 2096.4 2096.2 0.3T343-363 2212.1 2213.5 2212.3 1.4T338-357 2339.2 - 2339.0 0.2T73-101 2751.1 2751.8 2751.6 0.7T249-273 2800.5 2799.7 2799.2 1.3T1-24 2776.3 2819.4 2819.1 42.8 Acetylation at the initiator methionineT1-24 2776.3 - 2899.1 42.8�80 Phosphorylation of acetylated T1-24

T1-24 2776.3 - 2979.1 42.8�160 Double phosphorylation of acetylated T1-24

T338-363 2922.4 2922.8 2922.6 0.4T25-65 4595.1 4596.2 4596.2 1.1T25-65 4595.1 - 4676.5 81.4 Phosphorylation of T25-65

a Corresponds to the protonated monoisotopic mass except for those underlined. Underlined numbers are the protonated averagemass.

b �M is the largest difference between the predicted and observed mass, either control or irradiated.

858 Mass Spectrometric Analysis of p53 Modi®cations

phosphorylation site on p53 (Bischoff et al., 1990;Sturzbecher et al., 1990) and in vitro data demon-strating phosphorylation of Ser315 by cyclinA/cdk2 and cyclinB/cdc2 (Wang & Prives, 1995).

Discussion

In response to DNA damage, p53 mediates cellcycle arrest in G1 and G2 phases of the cell cycleprimarily through its ability to activate transcrip-tion of the cyclin-dependent kinase inhibitor, p21(Dulic et al., 1994; Stewart et al., 1995). Phosphoryl-ation of p53 at Ser315 by cyclinB/cdc2 or cyclinA/cdk2 was shown to stimulate the site-speci®c DNAbinding activity of p53 in vitro (Wang & Prives,1995). Because these cyclin/cdk complexes aremost prevalent in the S and G2 phases of the cellcycle (Pines, 1993), it was suggested that they maybe involved in regulating p53 activity, perhaps thatrequired for mediating cell cycle arrest in G2. We

have previously shown that p21 is induced inOCI/AML-3 cells exposed to radiation (Fu &Benchimol, 1997) and that these cells subsequentlyarrest in G1 and G2 (Sutcliffe et al., 1998). The con-stitutive phosphorylation observed at Ser315 inthis study makes it unlikely that phosphorylationat this site plays an important role in regulatingthe ability of p53 to promote cell cycle arrest inresponse to irradiation. This is the ®rst evidence ofphosphorylation at Ser315 using non-radioactivemeans. Previously, Price et al. (1995) demonstratedthat this site could be phosphorylated in vitro bycdk2. Using mutational analysis they also con-cluded that Ser315 was not involved in mediatingthe transactivation function of p53 and suggestedthat phosphorylation at Ser315 regulated someother function of p53.

While p53 protein levels are usually low in nor-mal cells, they are elevated following DNAdamage to mediate the growth suppressive func-

Figure 4. MALDI-TOFMS spectra of the p53 in-gel tryptic digests. MALDI-TOFMS analysis of in-gel tryptic digestof p53 bands isolated by 1D SDS-PAGE from immunopuri®ed extracts of (a) control and (b) irradiated OCI/AML-3cells. The spectra were acquired in positive ion, re¯ectron mode. Only the upper mass region of each spectrum is pre-sented (m/z 2000 > 5000). Approximately 10 % of each digest extract was used for this analysis. The matrix was dihy-droxybenzoic acid.

Mass Spectrometric Analysis of p53 Modi®cations 859

tions of p53. p53 protein levels are regulated bythe MDM2 protein which binds p53 at the amino-terminal region to promote ubiquitin-dependentdegradation of p53 (Haupt et al., 1997; Prives,

Figure 5. Identi®cation of phosphopeptides by nanoelectrspectra (normal acquisition, ÿve ion mode) obtained for thare presented in (a) and (b), respectively. The precursor ion sand irradiated samples are presented in (c) and (d), respectC18 ZipTips (Millipore) and eluted in 5 ml of 75 % methanoaverage of approximately 30 spectra acquired over ®ve minenergy was 180 eV for doubly deprotonated ions.

1998). DNA damage induced phosphorylation atSer15 and Ser20 of p53 results in dissociation ofbound MDM2 protein from p53 protein (Shiehet al., 1997; Siliciano et al., 1997; Unger et al., 1999a)

ospray ionization-precursor ion scanning. The full scane in-gel tryptic digests of the control and irradiated p53can (precursors of m/z 79, ÿve ion mode) for the controlively. The digests were desalted prior to analysis usingl, 0.05 % acetic acid. The precursor ion spectra are theutes. The collision gas was nitrogen and the collisional

Figure 6. MS/MS analysis of a phosphopeptide. Nanoelectrospray ionization MS/MS analysis of the in-gel trypticdigest of p53 isolated from control OCI/AML-3 cells. The precursor ion was m/z 711.1 and corresponds to the singlyphosphorylated tryptic peptide, T307-319 (MH2

2�). The amino acid sequence of this peptide is shown and the sites ofthe major fragmentations are also indicated. This spectrum is the average of approximately 60 scans acquired overtwo minutes. The collisional energy was 56 eV.

860 Mass Spectrometric Analysis of p53 Modi®cations

and implicates this modi®cation in stabilization ofp53 protein (Haupt et al., 1997; Kubbutat et al.,1997). While DNA-PK (Lees-Miller et al., 1992;Shieh et al., 1997), ATM (Khanna et al., 1998) andATR (Tibbetts et al., 1999) can phosphorylate Ser15in vitro, no kinase information is available forSer20. Mutational studies of phosphorylation siteshave yielded contradictory results regarding theirfunctional signi®cance (Ashcroft et al., 1999; Fuchset al., 1995; Blattner et al., 1999). Several studies,however, show that mutation of Ser15 or Ser20leads to reduced p53 transactivation function andgrowth suppressive capability (Unger et al., 1999b;Mayr et al., 1995; Fiscella et al., 1993). Ko et al.(1997) reported that phosphorylation at Ser33enhanced the site-speci®c DNA-binding activity ofp53 protein. In vitro studies show that while CAKphosphorylates Ser33 (Ko et al., 1997), DNA-PK(Lees-Miller et al., 1992; Shieh et al., 1997) and ATR(Tibbetts et al., 1999) phosphorylate Ser37.

We have used mass spectrometry to investigatethe phosphorylation and acetylation state of p53protein in untreated or irradiated OCI/AML-3cells. Our MALDI-TOFMS observation of IR-induced phosphorylation at the N terminus andacetylation at the C terminus of p53 is consistentwith reports that phosphorylation at the N termi-nus promotes acetylation of the C terminus follow-ing DNA damage. N-terminal phosphorylationwas shown to increase the binding of p53 proteinto acetyl transferases that acetylated the C termi-nus (Lambert et al., 1998; Sakaguchi et al., 1998).

Previous studies have relied on the use of 32P-labelling and phosphopeptide mapping. Thisapproach is not suitable for p53, since exposure toradioactive isotopes leads to stress-induction ofp53 by virtue of radiation damage from radioiso-tope decay (Yeargin & Haas, 1995; Bond et al.,1999). The use of phospho-speci®c and acetyl-speci®c p53 antibodies have provided an alterna-tive and non-radioactive means to identify speci®cresidues on p53 that undergo post-translationmodi®cation. The mass spectrometric analysisreported here is comprehensive and complemen-tary with these other approaches. Mass spectro-metric analysis of proteins, however, is notwithout certain drawbacks. We did not, forexample, identify all the predicted tryptic peptidesof p53 protein, including those from the Cterminus, which harbor several sites of phos-phorylation. Loss of peptides can occur for severalreasons: incomplete proteolytic digestion, incom-plete extraction from gel slices following digestionand loss during desalting due to the inability ofsome peptides to bind the C18 reverse-phase desalt-ing matrix. In addition, mass spectrometry ana-lyses have an inherent limitation in that theseprocedures are unable to recognize all peptides.Our study serves to con®rm previous phosphoryl-ation and acetylation studies and identi®es anadditional putative site at Lys319 for acetylationon p53.

Mass Spectrometric Analysis of p53 Modi®cations 861

Materials and Methods

Cells

All experiments were conducted with a cell line, OCI/AML-3, derived from the primary blasts of an AMLpatient (Wang et al., 1989). p53 cDNA from these cellswas sequenced and shown to be wild-type. There is aproline to arginine polymorphism at amino acid 72 (Fuet al., 1996). The cells were grown in suspension to a den-sity of about 0.6 � 106 cells per ml in alpha-MinimalEssential Medium (Gibco-BRL) supplemented with 10 %(v/v) fetal calf serum. Cells were irradiated in suspen-sion (or mock-irradiated), and returned to the incubatorfor two hours before lysis.

Purification of p53 protein

Immediately before OCI/AML-3 cells were harvestedfor lysis, inhibitors of phosphatases and kinases wereadded to give a ®nal concentration of 1 mM Na3VO4

and 50 mM NaF, to prevent changes in cellular phos-phorylation states during the centrifugation and lysisprocess. Cells were lysed in ice-cold modi®ed RIPAbuffer (150 mM NaCl, 10 mM Tris (pH 7.5), 1 % NP-40,1 % Triton X-100, 1 % (w/v) sodium deoxycholate, 0.1 %(w/v) SDS, 2 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 100 mg/ml Pefabloc (Boehringer Man-nheim), 0.05 mM Na2EDTA, 0.1 mM sodium vanadate,50 mM sodium ¯uoride, 10 mM sodium pyrophosphate).The lysates were sonicated to shear the DNA and centri-fuged to remove cellular debris. p53 protein in thelysates was puri®ed by af®nity chromatography usingPAb1801 cross-linked to Sepharose beads (Pharmacia).After application of the lysate, the beads were washedextensively with lysis buffer supplemented with 0.5 MNaCl. p53 was eluted with 5� column volumes of50 mM triethylamine (TEA), 150 mM NaCl, 0.1 % TritonX-100 (pH 11.5). The TEA eluate was immediately neu-tralized using 0.25 volumes of 1 M Tris (pH 6.8).Although we cannot be certain that base catalyzedhydrolysis of phosphates at some residues did not occurunder these conditions, the consistent presence of phos-phorylated p53 peptides in independent mass spec-trometry analysis indicated that there was no, orminimal loss of phosphates in our procedure.

p53 protein was puri®ed several times, and separatepreparations of puri®ed p53 protein were used for inde-pendent in-gel tryptic digestion and mass spectrometryanalyses.

Western analysis of p53 isoforms

Immunoaf®nity puri®ed p53 protein from control andirradiated OCI/AML-3 cells was subjected to electro-phoresis through a 10 % (w/v) or 8 % Tris-tricine gel(Gallagher, 1995). Following non-®xing silver staining(Shevchenko et al., 1996), the proteins in the p53 regionwere cut out as a series of consecutive thin bands andextracted by incubating the minced bands in 1� SDSsample buffer, and heating to 100 �C for ®ve minutes.The eluted proteins were separated by SDS-PAGE(9.5 %), electroblotted to PVDF membrane (Towbin et al.,1979) and probed for p53 with a polyclonal antibodyagainst p53, PAb7 (Oncogene Research).

SDS-PAGE, in-gel tryptic digestion andMALDI-TOFMS

Immunopuri®ed p53 protein (200 ng to about 500 ng)from OCI/AML-3 cells was resolved by gradient (5 % to20 %) SDS-PAGE. The bands were visualized by silverstaining (Shevchenko et al., 1996), or by zinc/imidazolereverse staining (Castellanos-Serra et al., 1996). Gel slicescontaining p53 were subjected to overnight in-gel trypticdigestion (Shevchenko et al., 1996). Brie¯y, the gel sliceswere dehydrated with acetonitrile and completely driedusing a centrifugal evaporator (Savant Speed Vac). Thegel pieces were rehydrated with 10 mM dithiothreitol(DTT) in 50 mM ammonium bicarbonate (pH 8.3) andincubated at 37 �C for one hour. This solution was thenreplaced with 50 mM iodoacetamide in 50 mMammonium bicarbonate (pH 8.3) and left in the dark atroom temperature for one hour. The gel slice was dehy-drated with acetonitrile and rehydrated with 50 mMammonium bicarbonate (pH 8.3) a number of times toensure that the gel was free of chemical reagents. The gelslice was completely dried and rehydrated in 20 ml of50 mM ammonium bicarbonate (pH 8.3) containing200 ng of modi®ed trypsin (Promega). Once this solutionwas fully absorbed by the gel slice, trypsin-free bufferwas added until the gel piece was just covered. Thesamples were incubated overnight at 37 �C.

The tryptic peptides were extracted from the gelpieces as follows. Following the digest, any solutionremaining extraneous to the gel slices was removed to afresh tube. The gel pieces were ®rst subjected to anaqueous extraction with sonication using 5 % acetic acid.The organic extraction of peptides from the gel sliceswas performed with 5 % acetic acid in 50 % acetonitrile,with sonication. The digest and extract solutions werecombined and evaporated to dryness. The peptideswere redissolved in 50 % acetonitrile, 5 % acetic acid(5 ml). The samples were analyzed by Voyager EliteSTR MALDI-TOFMS (Perspective Biosystems Inc.).Dihydroxybenzoic acid was used as a matrix and allspectra were acquired in positive ion, re¯ectron mode.About one-tenth of the total gel-extracted peptide samplewas used for this analysis.

Phosphopeptide identification and sequencing

In-gel digest samples were prepared in the mannerdescribed above and were reconstituted in 5 % aceticacid. The samples were desalted using ZipTipTM C18

reverse-phase desalting Eppendorf tips (Millipore). Thepeptides were trapped on the reverse phase media bypassing the digest solution back and forth through thetips a number of times. After rinsing the tips thoroughlywith 5 % acetic acid, the peptides were eluted with 75 %methanol, 0.05 % acetic acid and used for analyses byelectrospray mass spectrometry (ESMS).

Phosphopeptides were detected by precursor ion scan-ning (precursors of m/z 79) in negative ion mode ona API 300 triple quadrupole mass spectrometer(PE/SCIEX). A nanoelectrospray interface designed touse disposable glass capillary emitters (Micromass) wasbuilt in-house and installed on the instrument. Approx-imately 1-2 ml of desalted peptide solution was injectedinto the capillary. Precursor ion spectra were acquired inmultiple channel acquisition mode (MCA), typically overa period of ®ve minutes (m/z 300-2000, 0.5 mass unitstep size, 5 ms dwell time). Nitrogen was used as thecollision gas and the collision energy was 180 eV.

862 Mass Spectrometric Analysis of p53 Modi®cations

Peptide and phosphopeptide sequencing was achievedby tandem mass spectrometry (MS/MS) using a quadru-pole time-of-¯ight mass spectrometer (Q-TOF, Micro-mass) equipped with a nanoelectrospray ionizationsource. Usually, the same nanoelectrospray capillariesand desalted samples were used both for precursor ionscanning and MS/MS analysis. All MS/MS analyseswere carried out in positive ion mode. The nanoelectro-spray ionization voltage was 900-1100 V, the collisiongas was nitrogen and the collision energy was 50 to60 eV. MS/MS spectra were typically acquired everytwo seconds over a period of ®ve minutes. Approx-imately three-tenths of the gel-extracted peptide samplewas used for phosphopeptide identi®cation, and theremaining six-tenths for the MS/MS analysis.

Acknowledgments

We thank Dr Chris Richardson for the generous gift ofrecombinant p53 protein. We acknowledge with muchgratitude Dr Marees Harris-Brandt and John Shannonfor all their help. We are also grateful to Micromass foraccess to the Q-TOF instruments. This work was sup-ported by grants from the National Cancer Institute ofCanada and the Medical Research Council of Canada.

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Edited by M. Yaniv

(Received 13 September 1999; received in revised form 16 November 1999; accepted 26 November 1999)