Age-dependent modification of proteins: N-terminal racemization

Age-dependent modification of proteins: N-terminalracemizationBrian Lyons1, Ann H. Kwan2, Joanne Jamie3 and Roger J. W. Truscott4

1 Save Sight Institute, University of Sydney, Sydney Eye Hospital, NSW, Australia

2 School of Molecular Bioscience, University of Sydney, NSW, Australia

3 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

4 Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia

Keywords

ageing; aquaporin; human; long-lived

protein; racemization

Correspondence

R. Truscott, Illawarra Health and Medical

Research Institute, University of

Wollongong, Northfields Avenue,

Wollongong, NSW 2522, Australia

Fax: +61 2 4221 8130

Tel: +61 2 4221 3503

E-mail: [email protected]

(Received 21 November 2012, revised 19

February 2013, accepted 22 February 2013)

doi:10.1111/febs.12217

Age-dependent deterioration of long-lived proteins in humans may have

wide-ranging effects on health, fitness and diseases of the elderly. To a

large extent, denaturation of old proteins appears to result from the intrin-

sic instability of certain amino acids; however, these reactions are incom-

pletely understood. One method to investigate these reactions involves

exposing peptides to elevated temperatures at physiological pH. Incubation

of PFHSPSY, which corresponds to a region of human aB-crystallin that

is susceptible to age-related modification, resulted in the appearance of a

major product. NMR spectroscopy confirmed that this novel peptide

formed via racemization of the N-terminal Pro. This phenomenon was not

confined to Pro, because peptides with N-terminal Ser and Ala residues

also underwent racemization. As N-terminal racemization occurred at

37 °C, a long-lived protein was examined. LC-MS/MS analysis revealed

that approximately one third of aquaporin 0 polypeptides in the centre of

aged human lenses were racemized at the N-terminal methionine.

Structured digital abstract

• LAP cleaves AQP0 by enzymatic study (View interaction).

Introduction

Long-lived proteins are widespread in the human body

[1,2]. The most abundant are the collagens, which

account for ~ 20% of the body mass of an adult [3]

and have a half-life in the human body that is esti-

mated at 95 years [4]. Over time in a biochemical envi-

ronment, collagens and other persistent proteins such

as elastin [5,6], dentin [7,8], myelin basic protein [9,10]

and lens crystallins [11,12] undergo numerous changes.

Some of these have been characterized, for example

conversion of L-Asp and L-Asn residues into isomeric

D- and isoAsp forms via inter-molecular condensation

[13]. Spontaneous peptide bond cleavage at Asn resi-

dues of proteins may also occur via a succinimide

intermediate [14]. Such age-related modifications have

been implicated in provoking human autoimmune dis-

ease [15,16].

The human lens contains the highest protein concen-

tration of any tissue in the body, but there is no pro-

tein turnover [17]. This means that proteins in the

centre of the lens are present for a lifetime. This tissue

may therefore be used to examine post-translational

events that occur in other long-lived proteins. Promi-

nent modifications such as methylation [18,19], racemi-

zation [20–22], deamidation [13,20,23] and truncation

Abbreviations

AQP0, aquaporin 0; FT ICR, Fourier transform ion cyclotron resonance mass spectrometry; HSQC, heteronuclear single quantum coherence;

NOESY, nuclear Overhauser effect spectroscopy; PDA, photodiode array detector; TFA, trifluoroacetic acid; TOCSY, total correlation

spectroscopy.

1980 FEBS Journal 280 (2013) 1980–1990 ª 2013 The Authors Journal compilation ª 2013 FEBS

[24–26] have been characterized; however, it is likely

that others remain to be elucidated.

Some reactions of long-lived proteins may be mod-

elled simply by exposing proteins and peptides to

elevated temperatures [27,28]. In the present investiga-

tion, a peptide was exposed to heat at pH 7.4 and the

major products were characterized. A facile racemiza-

tion of the N-terminal amino acid residue was

observed, and a mechanism is proposed for its forma-

tion. This novel process may apply to all proteins with

unblocked N-termini, as analysis of aquaporin 0 from

older human lenses showed that ~ 30% of the N-ter-

minal methionine was present as the D- form. Conver-

sion of the terminal amino acid to the D- form may

assist in stabilizing long-lived proteins against degrada-

tion by aminopeptidases.

Results

Some reactions of long-lived proteins in the body may

be conveniently investigated by exposing proteins or

peptides to elevated temperatures under physiological

pH conditions. During incubation of a peptide, PFHS-

PSY, a major product (peptide X) was observed that

eluted after the PFHSPSY peak (Fig. 1). The amount

of this new component increased with duration of

incubation at 60 °C, as the amount of PFHSPSY

decreased (Fig. 2). In order to determine the structure

of peptide X, semi-preparative HPLC was performed

on a sample of PFHSPSY that had been incubated for

1 week. The collected peptide X was then examined by

NMR spectroscopy.

It was apparent that peptide X was closely related

in structure to the starting material. As peptide bonds

preceding Pro residues (i.e. Xaa-Pro, where Xaa is

any amino acid) may adopt one of two conforma-

tions (cis and trans) that may be simultaneously pres-

ent in solution because of the low energy barriers of

rotation about the peptidyl-Pro imide bond [29,30],

2D NMR spectroscopy was used to investigate

whether cis Xaa-Pro bond formation had occurred in

peptide X.

Spectroscopic identification of a cis Xaa-Pro peptide

bond by NMR traditionally relies upon observation of

a strong 1Ha–1Ha NOE correlation between the two

sequential residues [31]. In contrast, a trans Xaa-Pro

peptide bond is expected to show a 1Ha–1Hd NOE cor-

relation between the two sequential residues. If pep-

tide X is a cis Xaa-Pro isomer of PFHSPSY, there

should be a 1Ha–1Ha NOE correlation between Ser4

and Pro5. Analysis by 2D ROESY did not reveal any

such correlation, indicating it is unlikely that a cis Pro

bond is present.

As NOE correlations are prone to chemical shift

degeneration and artefacts originating from insufficient

water suppression, a 13C-HSQC spectrum was also

obtained. 13C chemical shifts are very sensitive to local

chemical environments, with 13Cb and 13Cc resonances

in Pro residues typically shifting up-field by 2–3 ppm as

the Xaa-Pro bond changes from trans to cis [32]. There

was a negligible change in the 13Cb and 13Cc chemical

shifts of the two samples; therefore, 13C chemical shift

analysis also suggests that peptide X is unlikely to be a

cis Pro variant of PFHSPSY (data not shown).

All NMR signals observed in the peptide X and

PFHSPSY spectra were unambiguously assigned to

atoms belonging to each amino acid residue in the

peptide sequence. Close examination revealed that the

NMR signals corresponding to one of the Pro residues

were shifted in peptide X compared with the starting

peptide (Fig. 3). The chemical shift values correspond-

ing to Pro1 in PFHSPSY and peptide X differed sig-

nificantly, whereas the signals corresponding to Pro5

in both peptides remained essentially the same.

This suggests that the N-terminal Pro (Pro1) is in a

different chemical environment in peptide X and is

likely to have undergone modification. However, the

fact that the change in chemical shifts was relatively

small in magnitude, and that neighbouring residues

were little affected, suggested that the modification

was localized.

As cis/trans isomerization of Pro did not appear to

explain the behaviour of the peptide, we suspected that

the N-terminal Pro may be racemized in peptide X. To

confirm this, acid digests of both PFHSPSY and pep-

tide X were examined by chiral HPLC to test for the

presence of D-Pro. Peptide X was found to contain

~ 50% D-Pro (Fig. S1). A (D-Pro)FHSPSY standard

was synthesized, and its 1H-NMR spectrum matched

that of peptide X (Fig. 4). In addition, the synthetic D-

Pro peptide co-eluted with peptide X by HPLC, as

indicated in Fig. 1.

Having established that N-terminal racemization

occurs in this peptide, other peptides were examined.

Two homologous heptapeptides with Ser and Ala as

the N-terminal residues were incubated and also found

to racemize at the N-terminus, although the rates of

racemization were lower than those of PFHSPSY

(Fig. 5A). Racemization should be an equilibrium

reaction, and this was established by incubation of

(D-Pro)FHSPSY under the same conditions as

PFHSPSY. Interestingly, the rate of the reverse reac-

tion, i.e. (D-Pro)FHSPSY to PFHSPSY, was lower

than that of conversion of the L-peptide to the D-Pro

form (Fig. 5B). A similar phenomenon was observed

with another peptide (MWELR) (Fig. S2).

FEBS Journal 280 (2013) 1980–1990 ª 2013 The Authors Journal compilation ª 2013 FEBS 1981

B. Lyons et al. N-terminal racemization of proteins

In order to determine whether racemization occurs

at physiological temperature, the incubation of PFHS-

PSY was repeated at 37 °C. As expected, the rate of

racemization of the N-terminal Pro in PFHSPSY was

lower (0.04 nmol�h�1 at 37 °C compared with

2.83 nmol�h�1 at 60 °C) (Fig. 6), but still reached lev-

els of ~ 5% after 10 weeks. Racemization of the N-ter-

minal Pro residue occurred at a similar rate in HEPES

(100 mM, pH 7.4) and TES buffers (100 mM, pH 7.4),

thus ruling out a major buffer effect. Decreasing the

pH using MES (pH 5.4) resulted in an approximately

sixfold reduction in the rate of N-terminal Pro

racemization at 60 °C (data not shown). A possible

role for metal ions was investigated by repeating the

incubation of PFHSPSY in phosphate buffer (pH 7.4)

with addition of 1 mM EDTA; however, N-terminal

racemization occurred at a similar rate to the control

(data not shown).

A

B

C

Fig. 1. HPLC traces showing elution of

PFHSPSY (A), the formation of peptide X

and other modified peptides following

incubation of PFHSPSY in phosphate

buffer (100 mM, pH 7.4) for 14 days at

60 °C (B), and the elution of (D-Pro)

FHSPSY (C). Detection at 280 nm. In

separate studies, SPSY was demonstrated

to form from FHSPSY, presumably via

diketopiperazine formation (see Fig. 8).

A modification involving the loss of the

N-terminal amino acid will be discussed

in a separate paper.


N-terminal racemization of proteins B. Lyons et al.

Having established that N-terminal racemization of

peptides occurs under physiological conditions, a long-

lived protein was examined to determine whether race-

mization was detected. Aquaporin 0, an abundant

integral membrane protein in the lens [33,34] was

chosen. Aquaporin 0 has a free N-terminal methionine

residue that is located within the cytosol. To investi-

gate N-terminal racemization of aquaporin 0, fibre cell

membranes from human lenses were isolated as

described previously [35] and then treated with trypsin.

The sequence of the N-terminal tryptic peptide of

human lens aquaporin 0 is MWELR [35]. Tryptic

digests were then analysed by two methods. First, sam-

ples of the digest were examined by semi-preparative

HPLC using conditions under which the peptide stan-

dards (L-Met)WELR and (D-Met)WELR were well

Fig. 2. Time course showing the loss of PFHSPSY and appearance

of peptide X at 60 °C.

Fig. 3. Partial 1H-TOCSY spectra of PFHSPSY (grey) and peptide X

(blue/green). The NMR signals were unambiguously assigned to

atoms belonging to each amino acid residue in peptide X and

PFHSPSY. Close examination revealed that the NMR signals

corresponding to Pro1 in PFHSPSY and peptide X differed

significantly, whereas the signals corresponding to Pro5 in both

peptides remained essentially the same.

A

B

C

Fig. 4. Comparison of partial 1H-NMR spectra of (A) PFHSPSY, (B)

peptide X and (C) synthetic (D-Pro)FHSPSY. Samples were pre-

pared in phosphate buffer (50 mM, pH 7.4) containing 1 mM

4,4-dimethyl-4-silapentane-1-sulfonic acid and 10% D2O.

A

B

Fig. 5. (A) N-terminal racemization of PFHSPSY, SFHSPSY and

AFHSPSY as a function of time. (B) Inter-conversion of (L-Pro)

FHSPSY and (D-Pro)FHSPSY. Each peptide was incubated in

phosphate buffer (100 mM, pH 7.4) at 60 °C.



resolved [(L-Met)WELR at 47.7 min and (D-Met)

WELR at 50.9 min].

Peaks from the lens digests eluting at the retention

times of (L-Met)WELR and (D-Met)WELR were col-

lected and examined by MALDI mass spectrometry.

Both the cortex (the outer, most recently synthesized,

part of the lens) and the nucleus (which contains pro-

teins synthesized pre-natally) of the human lenses were

analysed. For both lens regions, peaks at the elution

positions of (L-Met)WELR and (D-Met)WELR were

collected. The two HPLC peaks from the cortex and

the two HPLC peaks from the nuclear extracts were

collected and found to contain an ion at m/z 734.3.

This ion corresponds to the molecular ion of (L-Met)

WELR [or (D-Met)WELR]. MALDI MS/MS analysis

of each m/z 734 ion confirmed its identity by compari-

son with the MS/MS spectrum of synthetic (L-Met)

WELR (data not shown).

In the second method, ESI LC-MS/MS of unfrac-

tionated tryptic digests from pooled cortex and

nuclear fractions of five pairs of human lenses from

subjects aged 75–85 years was used to confirm the

identification, and to gauge the extent of racemiza-

tion more accurately. A selected ion monitoring trace

of the doubly charged molecular ions corresponding

to MWELR (368.6 [M+2H]2+) isolated from the lens

nuclear extract is shown in Fig. 7. The m/z 368.6

ion from each of the four peaks (i.e. the L- and

D-Met peptides from cortex and nucleus) was sub-

jected to MS/MS fragmentation and compared with

the MS/MS spectra of (L-Met)WELR/(D-Met)WELR

standards (Fig. S3). In each case, MS/MS analysis

confirmed the identity of the peptide as (L-Met)

WELR/(D-Met)WELR. Using the areas under the

curves, it was estimated that ~ 28% of the total aqu-

aporin 0 present in the nucleus of aged human lenses

is present as the D-Met form, whereas ~ 13% of

aquaporin 0 in the outer part of the lens had been

racemized.

Discussion

Long-lived proteins in the body undergo a number of

modifications during extended exposure to physiological

conditions [1,2,36]; however, details of several reactions

remain to be elucidated. In this study, it was demon-

strated that incubation of peptides at pH 7 results in fac-

ile racemization of the N-terminal amino acid. This was

observed at 37 °C, and the rate was increased at higher

temperatures. In reactions using model peptides, racemi-

zation was confined to the N-terminal amino acid resi-

due. One potential mechanism that may account for this

is shown in Fig. 8. This involves a central Schiff base

intermediate, which may decompose via one of two

pathways: one yielding a diketopiperazine and one pro-

ducing a racemized N-terminus.

Evidence in support of this mechanism comes from

the fact that two of the three peptide standards

(SFHSPSY and AFHSPSY) incubated at 60 °Cshowed significant racemization of the N-terminal resi-

due, with the racemization rate of N-terminal Ser

approaching that of Pro (Fig. 5A), and each also dis-

played some loss of an N-terminal dipeptide (data not

shown). Cleavage of the two N-terminal amino acids

of a peptide, linked together as a diketopiperazine, has

been described in detail by Steinberg and Bada [37].

It is proposed that racemization of the N-terminal

residue and loss of a cyclic diketopiperazine are com-

peting reaction pathways following formation of the

Schiff base, with steric factors determining the relative

formation of each. The case of PFHSPSY, for which

diketopiperazine formation was not detected, may be

considered an extreme example of this reaction. In this

case, N-terminal racemization is favoured almost

exclusively. It is also possible that other processes con-

tribute to the overall reaction pathway leading to loss

of the N-terminal amino acid, for example.

Therefore, racemization is not the only spontaneous

reaction that involves the N-terminal amino group. The

mechanism as outlined provides a pathway for forma-

tion of a racemized N-terminal amino acid, as well as

cleavage of the combined N-terminal and penultimate

residues as a diketopiperazine. A similar mechanism

was proposed by Sepetov et al. [38]. Although not all

amino acids were investigated as N-termini in this

study, those that were showed significant racemization.

Further evidence in support of this mechanism is shown

Fig. 6. N-terminal racemization of PFHSPSY at 37 °C. Samples

were incubated in phosphate buffer (100 mM, pH 7.4).



in Fig. 5B, where PFHSPSY with an L- form N-termi-

nal amino acid was found to racemize at a faster rate

than the corresponding D- form N-terminal amino acid.

If racemization occurred via simple abstraction and

re-addition of the a proton to the N-terminal amino

acid, these rates would be similar.

In addition, a stereo-specific difference in the rate of

inter-conversion was noted for another peptide, which

contained an N-terminal Met (Fig. S2). For both the

Pro and Met peptides, conversion rates to the other

isomer were higher when starting with the L-peptides

than the corresponding D-peptides (Fig. 5B). The rea-

son for this is not known, but presumably reflects con-

formational restrictions involved in formation of the

Schiff base intermediate and the degree to which it can

isomerize.

To determine whether N-terminal racemization is a

feature of long-lived proteins, aquaporin 0 from

human lenses was investigated. Lens proteins do not

turn over after their incorporation into mature fibre

cells, and are thus present for life [17,39]. Aquapo-

rin 0 is an integral membrane protein that acts as a

water channel and may also facilitate cell–cell interac-tions within the lens [40]. To study whether the N-ter-

minus of aquaporin 0 from adult lenses was

racemized, we took advantage of a technique used by

Schey et al. [35] that involves tryptic digestion of

intact lens membranes.

As the N-terminus of aquaporin 0, as well as the

C-terminus, are cytosolic [41], they are exposed in puri-

fied lens membrane fractions and may be selectively

proteolysed by enzymes such as trypsin. Analysis of the

nuclear region of aged lenses by LC-MS/MS revealed a

high level (28%) of racemization of the N-terminal

amino acid. A reduced amount of N-terminal racemiza-

tion was detected in the cortex (13%) (data not shown),

in accordance with the fact that nuclear proteins are, on

average, older than the cortical proteins [42].

This study demonstrated a novel modification

whereby the N-terminal residues of three peptides that

terminated in Pro, Ser and Ala all underwent

spontaneous racemization when incubated at pH 7.4.

Incubation of PFHSPSY at 37 °C was performed to

demonstrate that significant N-terminal racemization

A

B 100

Rel

ativ

e ab

unda

nce

90

80

70

60

50

40

30

20

10

030.0 30.5

30.28 30.89 31.57 31.98 32.56 32.83 33.0833.65

34.30 34.73 35.02 36.30 37.04 38.44 38.62 39.34 39.70

31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 38.5 39.0 39.5

Time (min)

100RT: 29.98 - 40.00 SM: 7G (L-Met)WELR

(D-Met)WELR

(L-Met)WELR

(D-Met)WELR

Rel

ativ

e ab

unda

nce

90

80

70

60

50

40

30

20

10

30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 38.5 39.0 39.5 40Time (min)

Fig. 7. ESI LC-MS selected ion monitoring trace of (A) a mixture of synthetic standards of (L-Met)WELR and (D-Met)WELR, and (B) a tryptic

digest of human lens membrane (nuclear fraction) derived from aquaporin 0. Detection at m/z 368.6.



(~ 5% over 10 weeks) occurred under physiological

conditions. Given the decades that some long-lived

proteins are exposed to in a similar environment, it is

proposed that this is a new post-translational modifica-

tion to which long-lived proteins with free amino

terminals may be subject. A biological example of this

modification (aquaporin 0) is provided, and work is

now underway to investigate whether this modification

is present in other long-lived proteins.

Racemization of internal amino acid residues such

as Asp/Asn [22,43] and Ser [44,45] in old proteins is

well known. Recent data suggest that racemization

rates at these sites are governed not only by the adja-

cent amino acid residue [13] but also by the structure

of the protein at the particular site [46]. Asp and Asn

residues in unstructured parts of crystallins are much

more susceptible to racemization than those present in

b-sheets [47].The ramifications of N-terminal racemization for

protein structure and function are as yet unknown,

and its impact will probably vary depending on the

particular protein. In terms of susceptibility to enzy-

matic proteolysis, it is very likely that having a

D-amino acid at the N-terminus will inhibit cleavage of

a long-lived protein by exopeptidases, as they are gen-

erally inactive on the D-isomers [48]. This was illus-

trated using the N-terminal peptide of aquaporin 0.

When exposed to leucine aminopeptidase, a protease

known to be widely distributed and present in the lens

[49], the L(Met) peptide was rapidly degraded whereas

the corresponding D(Met) peptide was stable (Fig. 9).

Some aminopeptidases are active on intact proteins

[50]. Leucine aminopeptidase appears to use peptides

as its major substrates, but may also have a low activ-

ity with intact proteins. It is not known whether ami-

nopeptidase degradation of long-lived proteins is a

physiologically relevant process.

In summary, racemization of the N-terminal amino

acid residue is a novel reaction that may be observed

in polypeptides that contain free a amino groups. It

joins an expanding array of reactions to which long-

lived proteins in the body are susceptible.

Fig. 8. Proposed mechanism to account

for racemization of the N-terminal amino

acid residue. Also shown is the pathway

leading to loss of the penultimate and

N-terminal amino acids together as a

diketopiperazine.



Experimental procedures

Materials

All peptides were synthesized by GLS Biochem (Shang-

hai, China). TFA (Sigma, St Louis, MO, USA) was of

spectrophotometric grade. Na2HPO4 and NaH2PO4 (high

purity) were purchased from Amresco (Solon, OH, USA).

Sequence-grade leucine aminopeptidase from porcine kid-

ney was purchased from Sigma. Trypsin (sequence grade)

was purchased from Promega (Madison, WI, USA). All

solutions were prepared in MilliQ water (Waters, Billerica,

MA, USA).

Peptide incubations

Peptides PFHSPSY, SFHSPSY, AFHSPSY and MWELR

were dissolved in triplicate in 100 mM phosphate buffer pH

7.4 (1 mg�mL�1) and incubated at 60 °C. PFHSPSY,

SFHSPSY and AFHSPSY are based on a region of

aB-crystallin (16–21) sequence but with varied N-termini

and with Tyr as the C-terminus. MWELR corresponds to

the N-terminal tryptic peptide of human aquaporin 0. A

drop of chloroform was added to each tube to prevent

microbial growth. Aliquots (20 lL) were taken at regular

time points and analysed by HPLC.

Peptidase incubations

Peptides (L-Met)WELR and (D-Met)WELT were incubated

(1 mg�mL�1) with 1 lg leucine aminopeptidase (1 : 000

enzyme : substrate) in 100 mM Tris buffer, pH 8.5, at

30 °C.

HPLC analysis and quantification

An Agilent 1100 HPLC system (Agilent Technologies, Santa

Clara, CA, USA) controlled using Chemstation software

and equipped with a PDA detector was used. Incubations

were monitored at 280 and 216 nm. Separation of the pep-

tides was achieved using a Jupiter Proteo 4 lm 90 �A column

(150 mm length, 4.6 mm internal diameter) at 40 °C. Thegradient was 0% B (0.1% TFA in acetonitrile) to 60% B

(0.1% TFA in acetonitrile) over 25 min. A standard curve

was generated for each peptide. The degree of racemization

was calculated based on the moles of each peptide formed as

a percentage of moles of peptide present at the start of the

incubation. The error bars refer to the standard deviation of

the replicates.

Semi-preparative HPLC purification

A Shimadzu Prominence HPLC system (Shimadzu, Kyoto,

Japan) controlled by Shimadzu Class VP software equipped

with a UV-vis detector (SPD-20A) and a fraction collector

(FRC-10A) was used. Purification of the peptides was

achieved using a Phenomonex Kinetex (Torrance, CA, USA;

100 mm length 9 4.6 mm internal diameter) 2.6 lm 100 �A

column at ambient temperature and was monitored at 280

and 216 nm. The gradient was 0% B (0.1% TFA in acetoni-

trile) to 60% B (0.1% TFA in acetonitrile) over 110 min.

Chiral amino acid analysis

Chiral amino acid analysis by HPLC was performed as

described by Goodlett et al. [51]. Peptides were hydrolysed

in 6 M HCl at 110 °C for 6 h, and then lyophilized.

ESI LC-MS/MS

Nano-liquid chromatography (nano-LC) was performed

using an Ultimate 3000 HPLC and autosampler system

(Dionex, Amsterdam, The Netherlands). Samples were

injected into a fritless nanoLC column (75 lm 9 10 cm)

containing Bruker (Billerica, MA, USA) C18 medium

(3 lm, 200 �A; Michrom) produced as described by Gatlin

[52]. Peptides were eluted using a linear gradient from 2%

B to 90% B over 37 min at a flow rate of 0.25 lL�min�1.

Mobile phase A consisted of 0.1% formic acid in H2O,

while mobile phase B consisted of acetonitrile : H2O (8 : 2)

with 0.1% formic acid. High voltage (1800 V) was applied

to a low-volume tee (Upchurch Scientific, Oak Harbor,

WA, USA), and the column tip was positioned ~ 0.5 cm

from the heated capillary (T = 250 °C) of a LTQ FT Ultra

mass spectrometer (Thermo Electron, Bremen, Germany).

Positive ions were generated by electrospray, and the

LTQ FT Ultra was operated in data-dependent acquisition

mode. A survey scan of m/z 350–1750 was acquired in the

FT ICR cell (resolution = 100 000 at m/z 400, with an

Fig. 9. Racemization of the N-terminus confers stability against an

exopeptidase. (L-Met)WELR and (D-Met)WELR were incubated

separately with leucine aminopeptidase, and aliquots were

removed for HPLC. Peptides were incubated in Tris buffer

(100 mM, pH 8.5) with leucine aminopeptidase (1 : 1000

enzyme : substrate).



accumulation target value of 1 000 000 ions). Up to six of

the most abundant ions (> 3000 counts) with charge states

> +2 were sequentially isolated and fragmented within the

linear ion trap using collisionally induced dissociation with

an activation q = 0.25 and activation time of 30 ms at a

target value of 30 000 ions. The m/z ratios selected for

MS/MS were dynamically excluded for 30 s.

NMR analysis

Samples were prepared in 50 mM phosphate buffer, pH 7.4,

containing 1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid

and 10% D2O. Spectra were acquired at 25 °C on an

Avance III 800 MHz NMR spectrometer (Bruker, Kar-

lsruhe, Germany) equipped with a triple-resonance TCI

cryoprobe. 1D 1H, 2D 1H-TOCSY, 2D 1H-ROESY and

2D 13C-1H HSQC experiments were acquired using stan-

dard Bruker pulse sequences.

Lens membrane digestion

Five lens pairs (from subjects aged 75–85 years) were dis-

sected into nuclear and cortical regions using a 6 mm

trephine. Tissues from the cortical regions were combined

as were the nuclear regions. Lens membranes were iso-

lated as described by Schey et al. [35]. In brief, each lens

region was homogenized in Tris buffer, pH 8.0, contain-

ing 6 M guanidine HCl to remove soluble and water-

insoluble proteins. Extracts were centrifuged at 16 000 g

(30 min, 4 �C) and the supernatants were removed. This

was repeated five times, then the pellets were homoge-

nized with water and centrifuged. This washing was

repeated five times, and the pellets were freeze dried. The

pellets were suspended in 50 mM ammonium carbonate,

pH 8.0, and 20 lg trypsin was added. Samples were

incubated at 37 °C for 16 h, and then freeze dried.

Acknowledgements

This study was supported by a grant from the

National Health and Medical Research Council

(NHMRC) (grant number 512334).

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Supporting information

Additional supporting information may be found in

the online version of this article at the publisher’s web

site:Fig. S1. A portion of the HPLC trace from chiral

HPLC analysis, comparing the acid hydrolysis prod-

ucts of PFHSPSY and peptide X.

Fig. S2. Percentage of (L-Met)WELR and (D-Met)

WELR that undergoes N-terminal racemization over

time.

Fig. S3. MS/MS spectra of (L-Met)WELR and (D-Met)

WELR synthetic standards, and (L-Met)WELR and (D-

Met)WELR peaks from the tryptic digest of a lens

nuclear membrane sample.



Age-dependent modification of proteins: N-terminal racemization

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