Detection and quantification of d -amino acid residues in peptides and proteins using acid hydrolysis

Accepted Manuscript Detection and quantification of D-amino acid residues in peptides and proteins using acid hydrolysis

Tetsuya Miyamoto, Hiroshi Homma PII: DOI: Reference:

S1570-9639(17)30300-X https://doi.org/10.1016/j.bbapap.2017.12.010 BBAPAP 40053

To appear in: Received date: Revised date: Accepted date:

20 October 2017 4 December 2017 19 December 2017

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ACCEPTED MANUSCRIPT

Detection and quantification of D-amino acid residues in peptides and proteins

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using acid hydrolysis

Tetsuya Miyamoto, Hiroshi Homma*

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Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane,

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Minato-ku, Tokyo 108-8641, Japan

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* Corresponding author.

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H. Homma, Tel: +81-3-5791-6229; Fax: +81-3-5791-6381;

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E-mail: [email protected]

Abbreviations

NMDA, N-methyl-D-aspartate; DLP-2, defensin-like peptide-2; DAEP, D-aspartyl endopeptidase; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; LC-MS/MS,

liquid

chromatography-tandem

1

mass

spectrometry;

HPLC,

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high-performance L-isoaspartyl

liquid

chromatography;

O-methyltransferase;

CID,

RP,

reversed-phase;

collision-induced

PIMT,

dissociation;

protein ECD,

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electron-capture dissociation; RDD, radical directed dissociation; IMS; ion mobility spectrometry; CHH, crustacean hyperglycaemic hormone; MALDI, matrix-assisted laser

desorption/ionisation;

VIH,

vitellogenesis

inhibiting

hormone;

NBD-F,

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4-fluoro-7-nitro-2,1,3-benzoxadiazole; ODS, octadecylsilylated silica gel; DCl,

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PT

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deuterium chloride; LHRH, luteinising hormone-releasing hormone

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Abstract Biomolecular homochirality refers to the assumption that amino acids in all

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living organisms were believed to be of the L-configuration. However, free D-amino acids are present in a wide variety of organisms and D-amino acid residues are also found in various peptides and proteins, being generated by enzymatic or non-enzymatic

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isomerization. In mammals, peptides and proteins containing D-amino acids have been

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linked to various diseases, and they act as novel disease biomarkers. Analytical methods capable of precisely detecting and quantifying D-amino acids in peptides and proteins

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are therefore important and useful, albeit their difficulty and complexity. Herein, we

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reviewed conventional analytical methods, especially 0 h extrapolating method, and the

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problems of this method. For the solution of these problems, we furthermore described

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our recently developed, sensitive method, deuterium-hydrogen exchange method, to detect innate D-amino acid residues in peptides and proteins, and its applications to sample ovalbumin.

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Keywords acid, Isomerisation, Racemisation, Acid hydrolysis, LC-MS/MS, Ovalbumin

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D-Amino

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1. Introduction Proteinogenic amino acids, except for glycine (Gly), can occur in two

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enantiomeric forms with D- or L-configuration. Amino acids in all living organisms were previously believed to consist exclusively of the L-configuration, and D-amino acid residues are exceptionally included in peptideglycans within bacterial cell walls

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and peptidic antibiotics [1–3]. Furthermore, recent progress in microanalytical and

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enantioseparation techniques has revealed free D-amino acids and D-amino acid residues in peptides and proteins in a wide variety of organisms including bacteria,

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plants and animals.

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Free D-serine (D-Ser) and D-aspartate (D-Asp) are found in various tissues and

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cells in mammals, including humans, at high concentrations, where they play important

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physiological roles. D-Ser is found in the brain, especially in the cerebral cortex and hippocampus, where it modulates neurotransmission upon binding as a coagonist to the Gly site of the N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor [4,5].

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The D-Asp concentration is maintained at high levels in the brain, pineal gland, pituitary gland and testis, and is associated with the regulation of hormonal secretion

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and steroidogenesis [6–15]. It was recently reported that D-glutamate (D-Glu) is metabolised in the mammalian heart by D-glutamate cyclase that converts D-Glu to 5-oxo-D-proline (5-oxo-D-Pro), although the physiological significance remains unclear

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at present [16].

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Additionally, D-amino acid residues in peptides and proteins have been studied [17,18], and found to result from both enzymatic and non-enzymatic post-translational

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isomerisation. A funnel spider venom, -agatoxin IV (with a D-Ser at position 46) [19],

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a cone shell venom, conotoxin (with D-Phe at position 44) [20], a platypus venom,

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defensin-like peptide-2 (DLP-2, with D-Met at position 2) [21], and a frog skin

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antibacterial peptide, bombinin H (with D-allo-Ile at position 2), are examples of the former [22]. These D-amino acid residues are produced by specific isomerases that act on the corresponding L-amino acid residue of precursor peptides or proteins. These D-amino

acid residues are critical for binding to the corresponding receptor and/or for

bioactivity.

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D-Amino

acid residues are also presumably produced by non-enzymatic

post-translational isomerisation, including in aged or diseased tissues [23,24]. For

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instance, D-Asp residues are found in specific sites of-crystallin in the lens [25,26], -amyloid peptide in the brain [27], and elastin in the aorta and skin [28–30]. Isomerisation of these Asp residues presumably occurs spontaneously via succinimidyl

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intermediates [23,24]. The resulting structural and functional changes in peptides and

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proteins are believed to cause various diseases such as cataract (isomerisation of -crystallin of the lens), Alzheimer’s disease (-amyloid peptide), and arteriosclerosis

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and elastosis (elastin in the aorta and skin). Therefore, the accurate determination and

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quantification of D-amino acids in proteins is essential for understanding the

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pathogenesis and treatment of age-related diseases.

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In this review, we describe the methods currently available for the detection and quantification of D-amino acid residues in peptides and proteins. Both amino acid sequence-dependent and -independent methods are summarised. In particular, we review conventional procedures based on sequence-independent methods, and explain

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the issues with the 0 h extrapolation method. We then focus on our sensitive method that

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solves this problem, summarise its applications to date and discuss future perspectives.

2. Methods for the detection and quantification of D-amino acids in peptides and proteins

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Available methods for the detection and quantification of D-amino acid

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residues in peptides and proteins are classified into two categories (Table 1): peptide or protein sequence-dependent methods (Fig. 1a), and sequence-independent methods (Fig.

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1b).

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In the case of sequence-dependent methods, a unique D-aspartyl

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endopeptidase (DAEP) is utilised that specifically cleaves at the C-terminal side of

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internal D-Asp residues in proteins. This enzyme can be of mammalian (DAEP) [31] and bacterial (paenidase) origin [32]. Using this enzyme, D-amino acid-containing proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [33]. Proteins treated with DAEP and untreated controls were separately by 2D-PAGE, and

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enzyme-treated spots disappeared, indicating that they may contain internal D-Asp residues. LC-MS/MS was then used for identification.

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Additionally, protein samples are usually first digested with proteinases (such as trypsin) for fragmentation of proteins. The resultant peptide fragments can be subjected to Edman degradation followed by chiral separation to determine the

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sequence and configuration of amino acid residues within the peptide [34–36]. The

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N-terminal phenylthiohydantoin amino acid that is cleaved from the peptide is separated using a chiral stationary phase, and racemisation of amino acids during the Edman

acids can be identified by high-performance liquid chromatography (HPLC)

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D-amino

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procedure must be avoided [35,36]. Alternatively, peptide fragments containing

acids display altered retention times relative to their corresponding all-L-amino

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D-amino

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[37–39]. Using reversed-phase HPLC (RP-HPLC), peptide fragments containing

acid fragments. After peptide separation, peak identification is carried out by comparison with synthetic peptide standards, by chiral separation of free amino acids following acid hydrolysis of the peptide or by determination of the molecular mass of peptide fragments by MS. Fujii et al. [40,41] identified four different peptide isomers

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containing Asp isomeric residues (L, L, D and D) using a combination of LC-MS and commercial enzymes. These unusual peptides are associated with aging and disease.

D-Asp

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The enzymes used for identification of isomeric residues were endoprotease Asp-N, endopeptidease (paenidase) and protein L-isoaspartyl O-methyltransferase

(PIMT). Peptides containing L-Asp and D-Asp residue are sensitive to endoprotease

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Asp-N and D-Asp endopeptidease (paenidase) digestion, respectively, whereas peptides

D-amino

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containing L--Asp are recognised and methylated by PIMT. acids in peptides can also be identified using MS/MS-based methods

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[42–50]. These methods are based on fragment ions generated upon ionisation that

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differ between peptides containing D-amino acids and their corresponding peptides

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comprising all-L-amino acids. A method using fragmentation by collision-induced

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dissociation (CID) was applied to terminal pentapeptides of -agatoxin and a tryptic dodecapeptide from amyloid -protein [42], and electron-capture dissociation (ECD) fragmentation was applied to tryptophan (Trp)-cage protein, lactoferrin peptide and dermorphin [43]. The combination of CID and radical directed dissociation (RDD) was applied to the analysis of crystallines extracted from eye lenses of some species [44,45].

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Ion mobility spectrometry (IMS) analysis of MS-generated fragment ions has been used for the identification of crustacean hyperglycaemic hormone (CHH) [46]. Furthermore,

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matrix-assisted laser desorption/ionisation (MALDI) is an efficient tool for discrimination of peptide isomers. MALDI coupled to tandem mass spectrometry (MALDI-TOF-TOF) was successfully applied to the identification of D-amino acid

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residues in dermorpin and a heptapeptide derived from vitellogenesis inhibiting

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hormone (VIH) [47], and the neuropeptide Asn-D-Trp-Phe-amide (NdWFa) [48], GdFFD [49] isolated from a sea slug, and peptide GH-2 from skin secretions of toad

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[50]. In addition, the determination methods of D-amino acid containing peptides using

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MS are carefully summarized by Jansson [51]. For sequence-dependent methods,

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standard peptides comprising all-L-amino acids are usually required. methods

using

specific

antibodies

against D-amino

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Immunological

acid-containing proteins can be applied to investigate their localisation in cells and tissues after protein sequence determination [52–56]. Fujii et al. prepared a highly specific antibody (Gly-Leu-D--Asp-Ala-Thr) for the detection of the D--Asp residue in A-crystallin in human lens [52] and lens-derived cell lines [54], and to detect

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D--Asp

in peptides in sun-damaged skin [29]. Soyez et al. [55,56] used a polyclonal

antibody with high specificity (pGlu-Val-D-Phe-Asp-Gln-Ala-Cys-Lys) to detect CHH

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in lobster and crayfish. In sequence-independent methods, peptide or protein samples are first hydrolysed under acidic conditions at high temperature, and the resultant free D- and acids are subsequently analysed by enantioseparation. Using this method, it is

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L-amino

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possible to determine the D-isomer content of all (19) proteinogenic amino acids in the samples. This method has been used for paleontological dating of ancient samples, and

acid content can also be investigated from hydrolysates of soluble fractions

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D-amino

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for age estimation of unidentified bodies using dental dentin samples [57,58]. The

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extracted from cells or tissues from eubacteria, archaea, fungi, plants and animals [59].

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In these studies, the detailed contents of D-amino acid residues were determined by use of 0 h extrapolation method.

3. Determination of D-amino acids by 0 h extrapolation As described above, the 0 h extrapolation method has been applied to

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determine the D-amino acid content following acid hydrolysis of peptides or proteins. The crucial feature of this method is the elimination of time-dependent racemisation of

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free amino acids during hydrolysis to facilitate the precise determination of the D-amino acid content.

We applied this method to determine the presence and content of D-amino

at

110C,

derivatising

the

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vapour

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acids in protein samples [60]. The method involves hydrolysis for 324 h under HCl

4-fluoro-7-nitro-2,1,3-benzoxadiazole

(NBD-F),

resultant

amino

separating

acids

derivatives

with on

a

on a chiral column. Racemisation of free amino acids released during

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L-enantiomers

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reversed-phase column and separating isolated NBD-amino acids into D- and

acid / total amounts of free D- + L-amino acids (D / (D + L)), against hydrolysis

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D-amino

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hydrolysis is corrected for by plotting the D-amino acid ratio, the amount of free

time, and extrapolating the linear regression line to 0 h to obtain the actual D-amino acid content from the y-intercept. Firstly, the D-amino acid content of commercially available free L-amino acids (Ala, Leu, Phe, Val, Asp and Glu) was determined in mock experiments by

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incubating at 110C under 6 M HCl vapour (standard conditions for protein hydrolysis), and values for D-amino acid content obtained by extrapolating the incubation time to 0

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h were close to those determined without incubation (Fig. 2a and Table 2) [60]. Subsequently, we investigated whether D-amino acid residues could be detected in purified -galactosidase using the 0 h extrapolation method. Recombinant

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-galactosidase was produced by E. coli and purified using a Ni column. The D-amino

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acid content (0.5−4%) was reproducibly determined for all amino acids tested (Fig. 2a and Table 2) [60]. Furthermore, the D-amino acid content (0.5−1.5%) was determined

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for the 40 residues human urocortin peptide produced by E. coli and purified in the same

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way (Table 2) [60]. The slope of the regression line reflects the rate of racemisation of

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each amino acid during hydrolysis (or incubation), and the slope of racemisation of free

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amino acids approximately matched the slope of racemisation during protein hydrolysis (Fig. 2a). This indicates that the effect of racemisation of free amino acids released from proteins by hydrolysis is adequately corrected for by extrapolating the linear regression line to 0 h. Therefore, the 0 h extrapolation value does not include the effect of racemisation of free amino acids, and hence likely reflects the D-amino acid content

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present in the original protein or peptide.

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4. Shortcomings of the 0 h extrapolation method Thus, it remains possible that D-amino acids can be artificially generated during the analytical procedure when determining the D-amino acid content using the 0

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h extrapolation method. We assumed that amino acid residues in peptides or proteins

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could undergo isomerisation before being cleaved during hydrolysis. In other words, 0 h extrapolation values can include contributions from such artificial D-amino acids (Fig.

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2b).

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We used two model dipeptides, L-Ala-L-Phe and L-Phe-L-Ala, to examine

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whether amino acid residues in peptides undergo isomerisation (conversion to

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diastereomeric peptides) during the early stages of acid hydrolysis, before being released as free amino acids followed by racemisation. These dipeptides were hydrolysed for short periods (0.5–2 h) at 110C under HCl vapour to trap the diastereomeric dipeptides. After hydrolysates were derivatised by NBD-F, they were separated into free amino acids (Ala and Phe) and diastereomeric dipeptides were

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isolated by HPLC with an octadecylsilylated (ODS) silica gel column. These diastereomeric dipeptides were subsequently separated into dipeptide enantiomers by

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HPLC with a chiral column to determine the content and proportion of each enantiomer [61].

The products from L-Ala-L-Phe hydrolysates included free Ala and Phe, the

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parent dipeptide L-Ala-L-Phe and the sequence-inverted dipeptide L-Phe-L-Ala [61].

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The amino acid sequence of a dipeptide incubated under acidic conditions can be easily inverted via formation of a diketopiperazine [62]. The formation of inverted dipeptides

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was observed during hydrolytic incubation, and the proportion of the inverted

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dipeptides was increased [61]. Furthermore, a small amount of diastereomeric dipeptide

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(D-Ala-L-Phe or L-Ala-D-Phe) was detected, and small quantities of diastereomeric

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sequence-inverted dipeptide (D-Phe-L-Ala or L-Phe-D-Ala) were also detected (Fig. 3a). Furthermore, to identify and quantify the dipeptide enantiomers generated

during acid hydrolysis, diastereomeric dipeptide fractions were separated into dipeptide enantiomers by HPLC with a chiral column. The generation of dipeptide enantiomers is summarised in Figure 3a. During hydrolysis of L-Ala-L-Phe under acidic conditions, the

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main product was L-Ala-D-Phe although both L-Ala-D-Phe and D-Ala-L-Phe were detected [61]. Furthermore, D-Phe-L-Ala and L-Phe-D-Ala were detected in the

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diastereomeric dipeptide fraction (as D-Phe-L-Ala or L-Phe-D-Ala) derived from the sequence-inverted dipeptide L-Phe-L-Ala (Fig. 3a). In addition, similar products were detected in L-Phe-L-Ala hydrolysates, among which L-Phe-D-Ala was the most

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abundant (Fig. 3a). These results indicate that amino acid residues in peptides undergo

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isomerisation (generation of D-amino acid residues) during the early stages of acid hydrolysis, before being cleaved into the free form.

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Additionally, the two model dipeptides L-Ala-L-Phe and L-Phe-L-Ala were

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hydrolysed for 3−24 h at 110°C under HCl vapour, and the D-Ala and D-Phe content in

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the parent peptides was determined by the 0 h extrapolation method (Fig. 3b). Values

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for Ala and Phe in L-Ala-L-Phe were 0.08% and 0.45%, respectively, and those for L-Phe-L-Ala

were 0.56% and 0.37%, respectively [61]. It follows that values for the

C-terminal amino acids were larger than those of the N-terminal amino acids for each dipeptide, even though the parent dipeptides consisted of only L-amino acids. This is consistent with the results of enantiomer analysis of dipeptide hydrolysates described

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above (Fig. 3b). Collectively, these results indicate that the contributions from artificial D-amino

acids in the 0 h extrapolation values were mainly derived from diastereomeric

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peptides generated during the early stages of acid hydrolysis, before being cleaved into the free form.

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5. Overview of the DCl hydrolysis method for determination of D-amino acids

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As described above, the 0 h extrapolation method is not sufficient for accurately determining D-amino acid residues in proteins, and another method is

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therefore needed to distinguish between authentic and artificial D-amino acids. The

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-hydrogen of amino acids associated with isomerisation of either free amino acids

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(racemisation) or peptidyl amino acids (epimerisation) is removed and rejoined during

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these processes, and therefore exchanges with hydrogen in the surrounding aqueous solvent. To label these exchangeable protons associated with isomerisation, Manning [63] developed a tritium-hydrogen exchange method in which hydrolysis is performed in tritiated HCl (3HCl), while Goodlett et al. [64] and Liardon et al. [65] developed a deuterium-hydrogen exchange method in which hydrolysis is performed in deuterium

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chloride (DCl). During hydrolytic incubation under DCl vapour, the molecular masses of artificial D-amino acids generated by racemisation or epimerisation increase by 1,

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since -hydrogen is substituted by deuterium, whereas those of authentic D-amino acids remain unchanged (Fig. 4). We established a sensitive detection system that combines DCl hydrolysis with LC-MS/MS using a chiral column. We analysed model peptides to

acids generated during hydrolysis [66]. The tripeptides L-Ala-L-Ala-L-Ala and

D-Ala-L-Ala-L-Ala

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D-amino

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confirm whether native D-amino acid residues could be distinguished from artificial

were hydrolysed at 110C under 6 M DCl vapour, hydrolysates were

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derivatised with NBD-F, NBD-amino acids were isolated by HPLC with an ODS

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column and samples were separated into D- and L-enantiomers on a chiral column and

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detected by MS/MS. The D-Ala content of L-Ala-L-Ala-L-Ala was 0% in the original

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molecular mass (Fig. 5a). By contrast, artificial D-Ala was detected as molecular mass + 1 (Fig. 5b). These results showed that innate D-Ala could be distinguished from artificial D-Ala generated during hydrolysis using this method. The D-Ala content of the D-Ala-L-Ala-L-Ala

was approximately 35% in the original molecular mass (Fig. 5c),

which is close to the theoretical value (33%). Furthermore, analysis of a decapeptide

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(D-Phe2,6-LHRH) derivative of luteinising hormone-releasing hormone revealed authentic D-Phe in the original molecular mass [66]. Therefore, we confirmed the

those generated during hydrolytic incubation.

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validity of this system to discriminate innate D-amino acids in model peptides from

As described above, D-amino acid residues were found to be present in

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recombinant -galactosidase produced and purified from E. coli cells using the 0 h

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extrapolation method. Thus, we used this sensitive method to examine whether recombinant -galactosidase contains innate D-amino acid residues [67]. In the original

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molecular mass, only the peaks of L-enantiomers were detected, while no peaks were

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observed for D-Ala, D-Leu and D-Phe [67]. Furthermore, peaks resulting from D-amino

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acids generated during hydrolysis were clearly detected as +1 molecular masses [67].

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Therefore, -galactosidase contains few innate D-amino acids, and the D-amino acid contents detected by the 0 h extrapolation method reflect D-amino acids generated artificially during hydrolytic incubation.

6. Application of our sensitive method for D-amino acid determination

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We applied our sensitive method to chicken ovalbumin, a major protein component of egg white, which was presumed to contain D-amino acid residues. During

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storage of unfertile eggs and the development of fertile eggs, ovalbumin is converted into the heat-stable S-ovalbumin form via the intermediary I-ovalbumin form [68−70]. The denaturation temperature of S-ovalbumin is approximately 8C higher than that of

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native ovalbumin [71,72]. The conversion of ovalbumin to S-ovalbumin under

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physiological conditions is associated with an increase in the pH of egg white to 9.5, which is caused by the release of carbon dioxide through the eggshell. Additionally,

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S-ovalbumin is artificially formed in vitro by alkaline treatment of native ovalbumin

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[69,70]. Although the mechanism for the thermostabilisation of S-ovalbumin has been

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extensively studied, the exact mechanism remains unclear [73–79]. Unique structural

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features of S-ovalbumin were revealed by X-ray crystallographic analysis [80], revealing three -sheets and 11 -helixes, similar to native ovalbumin. Interestingly, only three Ser residues (164, 236 and 320) out of 38 appeared to be in the D-configuration

(Fig. 6a). These serine residues are exposed to solvent at the surface of

the molecule, and analysis of ovalbumin mutants suggests that Ser164 and Ser320

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contribute to the thermostability of S-ovalbumin [81,82]. We investigated the generation of D-Ser residues in ovalbumin under mild

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alkaline conditions [83]. Native ovalbumin was incubated at pH 9.5 for approximately 10 days at 37C, followed by hydrolysis in DCl vapour, and determination of the D-Ser content by HPLC and LC-MS/MS. The D-Ser content of samples incubated at 37C

residues per molecule (Fig. 6b), in agreement with X-ray crystal structure

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D-Ser

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increased to 7.9% after 7 days, and ~8% between days 7 and 10, corresponding to three

analysis [80]. Additionally, the D-Ser content of control samples incubated at pH 9.5 for

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10 days at 0°C was 4.0%, and the D-Ser content of samples incubated at pH 7.4 for 10

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days at 37C was 1.7% [83]. These results indicate that isomerisation of serine residues

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in ovalbumin is enhanced by alkaline conditions and temperature. This research

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demonstrated that innate D-amino acid residues in proteins could be accurately determined using this analytical method. We subsequently confirmed the generation of D-Ser in wild-type (WT) recombinant ovalbumin and its S164V, S236G, S320V and S164V/S320V mutants, following incubation under mild alkaline conditions. The D-Ser content of the WT

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protein increased to approximately 8% after incubation at 37C for 9–12 days at pH 9.5, which appeared to be equivalent to native ovalbumin (Fig. 6c). However, the D-Ser

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content in the S164V, S236G and S320V mutants (5.8–7.0%) was lower than that in the WT protein (7.8%; Fig. 6c). In the S164V/S320V double mutant, the D-Ser content was markedly lower than that in both the WT protein and the single mutants (Fig. 6c).

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Collectively, these results suggest that three serine residues in ovalbumin undergo

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isomerisation. Interestingly, D-Val residues were not detected in WT or mutant proteins

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[83].

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7. Conclusion and future perspectives

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In this review, we described the methods currently used for the detection and

AC

determination of D-amino acid residues in peptides and proteins. In particular, we demonstrated that, using the 0 h extrapolation method that is widely employed at present, artificial D-amino acids generated during hydrolysis of samples cannot be precisely distinguished from innate D-amino acids in native proteins and peptides. We demonstrated how our recently developed sensitive method that combines deuterium

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labelling of the -hydrogens of amino acids with LC-MS/MS overcomes this problem. Isomerisation of amino acid residues (particularly Asp and Ser) in peptides and proteins

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is associated with aging and various diseases [23,24,84,85], and D-amino acid residue-containing peptides act as novel biomarkers of such diseases. Interestingly, isomerisation of amino acid residues in proteins is closely related to the surrounding

acid residues (D-Ala and D-Ser) in three different proteins (bovine carbonic

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D-amino

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environment to which the protein is exposed. Indeed, we demonstrated the generation of

anhydrase, hen egg lysozyme and -amylase) upon incubation under mild alkaline

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conditions (pH 9.5; Table 3) [83]. Ultraviolet irradiation can also induce isomerisation

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of Asp residues in proteins [29,86]. Furthermore, post-translational modification might

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facilitate the isomerisation of amino acid residues. The D-Ser content in native

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ovalbumin was significantly higher than that in recombinant ovalbumin following incubation at 0C (native, 4.0%; recombinant WT, 0.7%) [83]. Although the WT protein shares the same overall conformational properties as native ovalbumin [87,88], native ovalbumin undergoes post-translational modification [89,90]. In summary, D-amino acid residue-containing peptides and proteins appear to

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be widely distributed, and it is important to determine whether isomerisation is physiological or artificial (an artefact of the experimental procedures used for analysis).

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Our sensitive method is certainly useful for the identification of those peptide and proteins. Recently, this method has been approved and applied as more sensitive system that combines two-dimensional HPLC with MS/MS by Hamase et al. [91,92]. Indeed, it

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identified D-Asp and D-Pro in addition to D-Ser in samples stored under mild alkaline

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conditions [92]. Various amino acid residues in proteins may undergo isomerisation, as demonstrated by D-asparagine (D-Asn) in mouse lysozyme [93]. In addition, Livnat et al.

ED

[94] established the screening method for the identification of D-amino acid containing

PT

peptides. This method is composed of following three steps; digestion with

peptide

using

synthetic

peptides.

In

fact,

GdYFD

and

AC

containing

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aminopeptidase M, DCl hydrolysis, and confirmation of putative D-amino acid

SdYADSKDEESNAALSDFA were identified from Aplysia using this method. Therefore, it is now possible to exhaustively analyse and identify different D-amino acid residue-containing peptides and proteins in complex biological samples.

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Acknowledgements We are deeply grateful to Prof. Haruhiko Masaki, Department of

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Biotechnology, The University of Tokyo, for his generous support. We thank Prof. Kenji Hamase, Graduate School of Pharmaceutical Sciences, Kyushu University, for a fruitful

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research collaboration.

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Figure legends Figure 1 Flow charts for the determination of D-amino acid residues in peptides and

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proteins using (a) sequence-dependent and (b) sequence-independent analytical methods.

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Figure 2 D-Asp ratio during hydrolytic incubation of free L-Asp and -galactosidase.

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(a,b) Free L-Asp and -galactosidase were incubated for 3, 6, 16 and 24 h at 110C under 6 M HCl vapour. Mean values for two independent experiments for L-Asp

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(diamonds) and -galactosidase (squares) are plotted against incubation time with linear

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regression lines. (b) L-Asp residue in -galactosidase can undergo isomerisation before

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-galactosidase possibly include artificial D-amino acids. These figures are modified based on our previous report [60].

Figure 3 Generation of dipeptide enantiomers during acid hydrolysis of L-Ala-L-Phe and the origin of 0 h extrapolation values for L-Ala-L-Phe.

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(a) L-Ala-L-Phe is inverted to L-Phe-L-Ala by hydrolytic incubation. The inverted peptide, L-Phe-L-Ala, is epimerised primarily at the C-terminal residue, to L-Phe-D-Ala.

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Thus, L-Phe-D-Ala is inverted to D-Ala-L-Phe. Additionally, L-Ala-L-Phe is predominantly epimerised at the C-terminal residue, to L-Ala-D-Phe, although a small amount of D-Ala-L-Phe is generated. L-Ala-D-Phe is further inverted to D-Phe-L-Ala.

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Values in parentheses show the ratio of each enantiomer produced when L-Ala-L-Phe is

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incubated for 1 h under acidic conditions, and the size of each box represents the approximate proportion of diastereomeric peptides based on [61]. D-Phe is

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predominantly generated from L-Ala-L-Phe by acid hydrolysis. (b) L-Ala-L-Phe was

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incubated for 3, 6, 16 and 24 h at 110C under HCl vapour. Mean values for three

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independent experiments for Ala (diamonds) and Phe (circles) are plotted against

AC

incubation time with linear regression lines. The 0 h extrapolation values were derived from diastereomeric peptides generated during the early stages of acid hydrolysis. These figures are modified based on our previous report [61].

Figure 4 Deuterium labelling of artificial D-amino acids by DCl hydrolysis.

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During DCl hydrolysis of peptides and proteins, the molecular masses of artificial D-amino

acids generated by racemisation of free amino acids or epimerisation of

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peptides (dotted line) increase by 1 due to substitution of the-hydrogen for deuterium, while the molecular masses of innate D-amino acids remain unchanged.

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Figure 5 Multiple reaction monitoring (MRM) chromatograms for NBD-D-Ala and

MA

NBD-L-Ala isolated from hydrolysates of L-Ala-L-Ala-L-Ala and D-Ala-L-Ala-L-Ala. (a) NBD-L-Ala (m/z 253/236; MW+0) isolated from L-Ala-L-Ala-L-Ala hydrolysed for

ED

24 h. Authentic D-Ala was not detected in original masses. (b) NBD-D/L-Ala (m/z

PT

254/237; MW+1) isolated from L-Ala-L-Ala-L-Ala hydrolysed for 24 h. Artificial D-Ala

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was detected in original masses + 1. (c) NBD-D/L-Ala (m/z 253/236; MW+0) isolated

AC

from D-Ala-L-Ala-L-Ala hydrolysed for 24 h. Authentic D-Ala was detected in original masses. These figures are modified based on our previous report [66].

Figure 6 Structure of S-ovalbumin and the D-Ser content in native and recombinant ovalbumin incubated under mild alkaline conditions.

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(a) The 3D structure of S-ovalbumin (PDB ID: 1UHG). The backbone is shown in ribbon representation, and three D-Ser residues (164, 236 and 320) are shown in ball

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and stick representation. Native ovalbumin (b) and all recombinant ovalbumin proteins (c) were incubated at pH 9.5 for 1–12 days at 37C. Each protein sample was hydrolysed for 24 h at 110C under 6 M DCl vapour. The D-Ser content for Native

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(circles), WT (diamonds), S164V (squares) and S164V/S320V (triangles) proteins is

MA

plotted against incubation time. These figures were created based on our previous report

AC

CE

PT

ED

[83].

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Table 1 Methods for the detection and quantification of D-amino acid residues in peptides and proteins Sequence-dependent methods Identification method

Reference

DAEP

2D-PAGE and LC/MS/MS

[33]

containing proteins

Deltorphin

−

Synthetic -amyloid

−

Human A-crystallin

Trypsin

CHHs

Lys-C

VIH

Asp-N

Human lens proteins

HCl hydrolysis, Edman degradation and HPLC Edman degradation and HPLC

[34]

[36]

RP-HPLC

[37]

HCl hydrolysis and RP-HPLC

[38]

RP-HPLC

[39]

Trypsin, Asp-N, DAEP and PIMT

RP-HPLC and LC/MS/MS

[40]

−

MS/MS (CID)

[42]

MA

ED

LC/MS/MS (CID and ECD) LC/MS/MS (CID and RDD)

−

PT

Pentapeptides of -agatoxin and tryptic dodecapeptide of amyloid -protein Trp-cage protein, lactoferrin peptide and dermorphin Crystallins from eye lenses of some spices

SC RI PT

D-Asp

Enzymatic digestion

NU

Peptide or protein

Trypsin

[43] [44] [45]

−

LC/MS/MS-IMS

[46]

Dermorpin and heptapeptide derived from VIH

−

MALDI-TOF-TOF

[47]

NdWFa

−

MALDI-TOF-TOF

[48]

GdFFD

−

MALDI-TOF-TOF

[49]

Peptide GH-2

−

MALDI-TOF-TOF

[50]

Peptide or protein

Degradation

Chiral analysis

Reference

Mollusc shells

HCl hydrolysis

Gas chromatography

[57]

Human teeth (dentin)

HCl hydrolysis

Gas chromatography

[58]

AC

CE

CHHs

Sequence-independent methods

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HCl hydrolysis

RP-HPLC

[59]

-Galactosidase

HCl hydrolysis

RP-HPLC

[60]

AC

CE

PT

ED

MA

NU

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Soluble fractions extracted from the cells or tissues

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Table 2 The D-amino acid content of free L-amino acids and 0 h extrapolation values for free L-amino acids, -galactosidase and urocortin D / (D + L)

(%)

Glu

Leu

Phe

Ala

Val

0

0.63

0

0.02

0.01

0.02

Mockb

0.12

0.65

0.01

0.04

0.03

0.11

-Galactosidaseb

1.74

2.08

1.63

4.19

1.77

0.56

Urocortinb

1.53

1.17

0.99

1.42

0.98

0.58

a

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Measurementa

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Asp

The D-amino acid content was determined without incubation at 110C under 6 M HCl

b

MA

vapour. Mean values for four independent experiments are presented. The D-amino acid content was determined by plotting each D-amino acid ratio against

ED

the reaction time at 110C under 6 M HCl vapour and extrapolating the linear regression line to 0 h. Mean values for two independent experiments are presented. For urocortin,

PT

mean values for six independent experiments are shown.

AC

CE

This table was created based on our previous report [60].

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Table 3 The D-amino acid content of carbonic anhydrase, lysozyme and -amylase incubated under mild alkaline conditions D / (D + L)

Carbonic anhydrase

-Amylase

Lysozyme

Ser

Ala

3

n.d.

n.d.

n.d.

5

0

0.8

0

6

n.d.

n.d.

n.d.

12

0.2

4.8

0

Ser

Ala

Ser

n.d.

0

0

0.6

n.d.

n.d.

n.d.

0.5

1.6

0.6

n.d.

n.d.

NU

Ala

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Days

(%)

n.d. = not determined.

MA

Carbonic anhydrase was from bovine erythrocytes, lysozyme was from hen egg white and -amylase was from porcine pancreas.

AC

CE

PT

ED

This table was created based on our previous report [83].

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Highlights • Methods for detecting and quantifying D-amino acids in polypeptides are reviewed

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• Existing methods are divided into sequence-dependent and -independent categories

• Problems with the 0 h extrapolation method based on acid hydrolysis are explained

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• We review our novel, sensitive method for detecting innate D-amino acids

AC

CE

PT

ED

MA

• We review recent applications of our novel method

52

Graphics Abstract

(a) Sequence-dependent method

(b) Sequence-independent method Degradation

Figure 1

(a)

(b)

10

5

Asp

Asp

β-Galactosidase

4

β-Galactosidase D/(D+L) (%)

D/(D+L) (%)

8 6

Free L-form 4 2

Isomerization

3 2

Free L-form

1

0

0 0

10

20

30

Hydrolysis time (h)

0

2

4

Hydrolysis time (h)

Figure 2

6

8

(a)

(b)

LALF

LFLA (30.121%)

(69.400%)

LADF (0.294%)

DFLA

LFDA

(0.103%)

(0.068%)

D/(D+L) (%)

1

0 0

2

4

6

Hydrolysis time (h) D AL F (0.014%)

Figure 3

8

H 2N

H

O

C

C

OH + H2N

N H

O

C

C

R

N H

H

O

C

C

R

H 2N

O

C

C

OH

MW + 0

OH

MW + 1

R

R

H

H

D

O

C

C

OH + H2N

D

O

C

C

R

R

DCl hydrolysis

N H

Figure 4

D

O

C

C

R

N H

D

O

C

C

R

Figure 5

Figure 6