De Novo Sequencing of Tryptic Phosphopeptides Using Matrix

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De Novo Sequencing of Tryptic Phosphopeptides using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment Daiki Asakawa, Hidenori Takahashi, Shinichi Iwamoto, and Koichi Tanaka Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04635 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Analytical Chemistry

De Novo Sequencing of Tryptic Phosphopeptides using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment

Daiki Asakawa,1* Hidenori Takahashi,2, Shinichi Iwamoto,2 and Koichi Tanaka2

1. National Institute of Advanced Industrial Science and Technology (AIST), National Metrology Institute of Japan (NMIJ), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8568, Japan 2. Koichi

Tanaka

Mass

Spectrometry

Research

Laboratory,

Shimadzu

Corporation,

1

Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604-8511, Japan

Correspondence to: Daiki Asakawa National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba Central 2, Umezono 1-1-1, Tsukuba, Ibaraki, 305-8568, Japan TEL: +81-29-861-0586, E-mail: [email protected]

ORCID: Daiki Asakawa (0000-0002-9357-8420) Hidenori Takahashi (0000-0001-6887-1724)

Keywords;

guanidination, phosphorylation site,

N–Cα bond cleavage,

protonation site,

collision-induced dissociation

ACS Paragon Plus Environment 1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS/MS) with radical-based fragmentation techniques has proven to be a promising method for phosphoproteomic applications, owing to its ability to determine phosphorylation sites on proteins. The radical-induced fragmentation technique involves the attachment or abstraction of hydrogen to peptides in an ion trap mass spectrometer, in a process called hydrogen attachment/abstraction dissociation (HAD), which has only been recently developed. In the present investigation, we have analyzed model phosphopeptides and phosphoprotein digests using HAD-MS/MS, combined with matrix-assisted laser desorption/ionization (MALDI), in order to demonstrate the usefulness of the HAD-MS/MS-based analytical method. The tryptic peptides were categorized as arginine- and lysine-terminated peptides and MALDI HAD-MS/MS is found to facilitate the sequencing of arginine-terminated tryptic peptides, because of the selective observation of C-terminal side fragment ions. In contrast, MALDI HAD-MS/MS of lysine-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. The guanidination of peptide converted lysine into homoarginine, which facilitated the interpretation of MALDI HAD-MS/MS mass spectra. The present method was useful for de novo sequencing of tryptic phosphopeptides.

Graphic Abstract


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INTRODUCTION The reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) side chain residues is the most abundant regulatory, covalent protein modification.1 Protein phosphorylation and dephosphorylation are involved in many biological processes, including signal transduction, cell division, gene expression, cytoskeletal regulation, and metabolic maintenance.1-2 Therefore, the characterization of protein phosphorylation is critically important, and it is usually performed by mass spectrometry. For the characterization of proteins, proteins are usually reduced, alkylated (e.g., S-carbamidomethylation), and finally, enzymatically digested to generate peptides. These peptides are then ionized by mass spectrometry to establish a peptide mass fingerprint that can be compared to the “in silico” digestion of proteins from databases, with the aim of identifying the digested protein. This analytical work flow is available only for protein identification, because the database is based on the DNA sequence, which has information about the primary sequence of the protein. Because post-translational modifications are not predicted from “in silico” protein databases, they should be directly characterized by mass spectrometry. In particular, de novo sequencing using tandem mass spectrometry (MS/MS) is essential for the determination of the site of post-translational modification, such as protein phosphorylation. Typically, amino acid sequencing of protein is performed by MS/MS with collision-induced dissociation (CID).3-4 Because the phosphate ester bond is labile, CID of phosphopeptides results in the dominant loss of the phosphoric group (80 and/or 98 Da) from the precursor ions; this loss of the phosphoric group can then be used as a diagnostic ion for phosphorylated peptide identification. However, the loss of phosphoric acid(s) through CID is unfavorable for determining the location of phosphorylated sites. Alternative to CID, electrospray ionization (ESI)-based tandem mass spectrometry with radical fragmentation, involving the electron association of multiply-charged peptides, such as electron capture dissociation (ECD)5 and electron transfer dissociation (ETD)6, has been used for phosphopeptide sequencing.7-9 Regarding the ECD/ETD mechanism, electron attachment/transfer


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occurs competitively at positively charge sites10 and π* antibonding orbital of peptide bond11-13 in a multiply-charged peptide, producing an aminoketyl radical intermediate. The produced radical intermediate immediately undergoes the cleavage at N–Cα bonds of the peptide backbone. One of the main advantages of ECD/ETD is that it gives rise to fragment ions, because of the N–Cα bond cleavage, without abundant fragmentation, allowing the determination of the phosphorylation site location.8,

14-15

Importantly, employing ions with a higher charge state as the precursors for

ECD/ETD dramatically improved the sequence coverage by increasing the yield of reactive radical species.16-18 However, tryptic peptides were mainly detected in the doubly-charged form, which does not provide enough sequence information from ETD-MS/MS. As a consequence, ETD-MS/MS has not yet become the method of choice for large-scale phosphopeptide analysis, owing to the difficulty of producing tryptic phosphopeptides with more than three positive charges by ESI. Regarding the peptide radical formation, CID of Cu2+-ligand-phosphopeptide complexes has been demonstrated to generate the peptide radical cation.19 CID of phosphopeptide radical cation provide both backbone cleavage and phosphoric acid loss, suggesting that the presence of radical facilitates the peptide backbone fragmentation.19 The photodissociation is also used for peptide radical formation. Phosphorylation sites can be modified to photoreactive group through β-elimination followed by Michael addition. Photodissociation of the modified peptides selectively induces homolytic cleavage at the modification site, generating a peptide radical.20-21 The obtained peptide radical undergoes peptide backbone fragmentation at the previously phosphorylated residue to allow the identification of phosphorylation site.20-21 A number of studies have been demonstrated the usefulness of tandem mass spectrometry with radical fragmentation for the analysis of peptides with post-translational modification.8, 22 Similar to ECD/ETD, the radical fragmentation occurring at ion source of matrix-assisted laser desorption/ionization (MALDI) mainly produced fragment ions due to N–Cα bond cleavage without the degradation of post-translational modifications. This technique is called MALDI in-source decay (ISD), and the choice of matrix is of critical importance for MALDI-ISD



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experiment.23 Briefly, the use of a reducing MALDI matrix leads to formation of an aminoketyl radical by hydrogen-atom transfer from the matrix to the analyte,24-26 and the resulting radical then undergoes the N–Cα bond cleavage located on the C-terminal side of the radical site27-29. In contrast to reducing matrix, the use of an oxidizing matrix induces Cα–C bond cleavage through a hydrogen-deficient peptide radical.30-31 Importantly, MALDI-ISD fragmentation occurs in the ion source, and it does not allow for precursor ion selection. Therefore, the most promising application of MALDI-ISD is top-down sequencing of purified proteins, including site determination of post-translational modifications.32-33 However, MALDI-ISD could not be applicable for the analysis of phosphopeptides in a tryptic digest mixture.34 Recently, fragmentation techniques involving the interaction between peptide ion and hydrogen atom in gas phase, named hydrogen attachment/abstraction dissociation (HAD), have been developed by Takahashi and co-workers.35 As in the case of MALDI-ISD, HAD mainly leads to N– Cα bond cleavage of the peptide backbone, without degradation of post-translational modifications, and has been utilized for the sequencing of modified peptides. HAD is initiated by hydrogen radical attachment to the peptide ion in the ion trap, and then the resulting aminoketyl radical intermediate undergoes N–Cα bond cleavage. In contrast, alternative cleavage at the Cα–C bonds of the peptide backbone occur as a minor process in HAD, through the hydrogen-deficient peptide radical formed by hydrogen abstraction. Briefly, HAD proceeds through hydrogen-abundant and hydrogen-deficient radical intermediates that share some mechanistic similarities with MALDI-ISD with reducing and oxidizing matrices, respectively. In contrast to MALDI-ISD, HAD is a fragmentation technique that occurs in the ion trap and allows for precursor ion selection. Therefore, HAD should be applicable for the analysis of a digested protein mixture. In this investigation, we have analyzed tryptic phosphopeptides by MALDI HAD-MS/MS. Although HAD of lysine (Lys)-terminated tryptic peptides showed complex mass spectra, the use of guanidination of Lys residues facilitated tryptic peptide sequencing by HAD-MS/MS.


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EXPERIMENTS Materials O-methylisourea,

α-casein,

α-cyano-4-hydroxycinnamic

acid

(CHCA)

and

3-aminoquinoline (3-AQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium dihydrogen phosphate aqueous solution (1 M) and ammonia aqueous solution (10 % wt) were purchased from Wako pure chemical (Osaka, Japan). The enzymes, trypsin (sequence grade modified trypsin, from porcine pancreas) and lysylendopeptidase (Achromobacter protease I) were purchased from Promega (Madison, WI, USA) and Wako pure chemical, respectively. Synthetic peptides and phosphopeptides were designed to mimic tryptic digestion of yeast enolases; peptides T18p (NVPLpYK), T19p (HLADLpSK), T43 (VNQIGTLSESIK), T43p (VNQIGpTLSESIK), and T43pp (VNQIGTLpSEpSIK) were purchased from CS Bio Co., Ltd. (Shanghai, China). The detailed peptide information is shown in Table. 1. All reagents were used without further purification. All the solvents used were of HPLC-grade quality, except for water, which was purified using a Milli-Q purification system (Millipore; Billerica, MA, USA).

Table 1. Monoisotopic Mass (Mm), Sequence, and Composition of Analyte Peptides Used. Peptide

Mm

Sequence

Composition

T18p

812.383

NVPLpYK

C35H57O12N8P

T19p

862.394

HLADLpSK

C34H59O14N10P

T43

1287.703

VNQIGTLSESIK

C55H97O20N15

T43p

1367.670

VNQIGpTLSESIK

C55H98O23N15P

T43pp

1447.635

VNQIGTLpSEpSIK

C55H99O26N15P2

Tryptic Digestion of Proteins The phosphorylated protein, α-casein was digested with trypsin and a lysylendopeptidase



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mixture. Each of the analyte proteins was dissolved in 25 mM ammonium bicarbonate aqueous solution at a concentration of 1 mg/mL (approximately 50 µM). A total of 0.5 µg each of lysylendopeptidase and trypsin were added to 100 µL of protein solution, followed by incubation at 37°C for 15 h.

Guanidination of Tryptic and Model Peptides The Lys residues in the analyte peptides were guanidinated according to a previously published protocol.36 An O-methylisourea aqueous solution was prepared at a concentration of 1 g/mL. The guanidination reaction mixture was prepared by mixing 10 µL of tryptic digest (approximately 50 µM) or 5 µL of model peptide (100 µM) with 10.5 µL of 4 N ammonia and 1.5 µL of the O-methylisourea aqueous solution, followed by incubation at 65°C for 10 min. The reaction was terminated by adding 15 µL of 10% formic acid aqueous solution (v/v). The guanidinated peptides were purified using GL-Tip™ SDB (GL Science, Tokyo, Japan) and then eluted with 50 µL of water/acetonitrile (50/50, v/v), containing 0.1 % formic acid. The final concentration of the analyte peptides was approximately 10 µM, as estimated from the amount of peptides/proteins used.

Preparation of Peptide Sample for MALDI Analysis A liquid matrix, 3-AQ/CHCA, was prepared according to a previously published protocol.37 Briefly, CHCA was dissolved in a methanol to give a saturated solution. 7 mg of 3-AQ was dissolved in 30 µL of the saturated CHCA methanol solution and 3 µL of 100 mM ammonium dihydrogen phosphate aqueous solution was then added to 3-AQ/CHCA solution. 0.5 µL of the analyte peptide (approximately 10 µM) and 0.5 µL of 3-AQ/CHCA solution were deposited on the MALDI plate. The peptide amount in each spot is estimated to be 5 pmol.

MALDI-HAD Tandem Mass Spectrometry


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MALDI-HAD-MS/MS experiments were performed using a prototype MALDI QIT-TOF mass spectrometer, based on the design of the AXIMA Resonance (Shimadzu/Kratos, UK) equipped hydrogen radical source. The details of the instrument construction were described in the previous report.35 Briefly, the H• was generated by passing H2 gas through a heated tungsten capillary and introduced into the ion trap through an aperture in the ring electrode. Analyte ions were generated by irradiation with a nitrogen laser (wavelength 337 nm) and were subsequently transported into the ion trap through an aperture on an endcap electrode. The trapped ions were cooled by the collision with helium. For MS/MS experiment, argon and hydrogen atom were used for CID and HAD, respectively. The trapped ions were extracted with an accelerating voltage of 10 kV, and ions were detected by a reflectron time-of-flight mass spectrometer. The laser power was optimized to obtain MALDI mass spectra that had high signal-to-noise ratios for the analyte ion peaks. Total MALDI-MS and subsequent HAD-MS/MS spectra were obtained by the accumulation of 50 and 100 laser shots, except for MS/MS analysis of the α-casein tryptic digests. For the MS/MS spectra of the α-casein tryptic digests, 50 and 200 laser shots were acquired for CID-MS/MS and HAD-MS/MS experiments, respectively. Notably, the yield of hydrogen atom is dependent on the temperature of heated tungsten capillary, which is controlled by electric current passing through the surrounding heater wire. As demonstrated previously35, the reaction time can be shortened below 0.5 s, when current value was up to 15 A (approximately 240 W). In this condition, the heated tungsten capillary also emit thermal electrons, which contributes to increase background level in the low m/z region. The fragments in the low m/z region might be hidden by the interference of background signals due to thermal electrons. To avoid interfering of thermal electrons, HAD-MS/MS experiment is operated with lower electric current (13 A, 170 W). As a result, the reaction time in the present study was set to 2.5 s. The interfering of thermal electrons can avoid by optimization of instrument configuration, and thereby the reaction time for HAD can be shortened in the near future.



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Notation In the present study, Zubarev’s unambiguous notation was adopted for peptide fragment ions.38 According to this notation, homolytic N–Cα bond cleavage yields the radical c• and z• fragments, and addition of a hydrogen atom to the c• or z• fragments produces a c' or z' fragment, respectively. The abstraction of a hydrogen atom from the c• or z• fragments produces a c or z fragment, respectively. Peptides annotated with an asterisk (peptide*) correspond to the peptide containing guanidinated Lys residue, (peptide + 42.02 Da).

RESULTS AND DISCUSSION Peptide T43 (VNQIGTLSESIK) and the phosphorylated forms, T43p (VNQIGpTLSESIK) and T43pp (VNQIGTLpSEpSIK), were analyzed by MALDI with HAD-MS/MS, as the tryptic peptide models. MALDI produced singly protonated peptides, which were then used as the precursors for HAD-MS/MS experiments. Figure 1 shows the HAD-MS/MS spectra of singly protonated T43, T43p, and T43pp. As described above, the HAD produced frangible radical intermediate, which leads to the selective cleavage at the N–Cα bond of the peptide backbone. Although CID fragmentation of protonated peptides involves intra-molecular proton migration,39 HAD induced N–Cα bond should occur independent of proton location. Therefore, the protonation site in peptide would not change during HAD processes and the observed fragment ions in the HAD-MS/MS spectrum is depend on the position of the protonation site in the peptide. In the case of the model tryptic peptides, the basic amino groups are present at the N-terminus and the Lys residue at the C-terminus; thereby, HAD of model peptides would give both N- and C-terminal positive fragment ions. As expected, figure 1 showed c' and z' series ions, accompanied by the weak signal of x' and y' ions. The x' and y' fragments would be formed by hydrogen abstraction from the peptide and a CID-like process, respectively. As described above, HAD mainly induced N–Cα bond cleavage through a aminoketyl radical intermediate, without degradation of the phosphate group. Therefore,


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HAD-MS/MS is a potentially useful method for the determination of the phosphorylation site location in tryptic peptides. Notably, peptide sequencing by HAD-MS/MS is generally conducted by interpreting mass differences between a series of consecutive c' and z' ions. As shown in figure 1, HAD of Lys-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. As a result, the presence of both c' and z' ions in the HAD-MS/MS spectrum made the attribution of m/z values to a specific series of consecutive c' and z' ions difficult.

Figure 1. HAD-MS/MS mass spectra of peptides, (a) [T43+H]+, (b) [T43p+H]+, and (c) [T43pp+H]+. Asterisks indicate precursor ions.

As described above, the Lys-terminated tryptic peptides gave both N- and C-terminal side fragments in the HAD-MS/MS spectrum, because of the presence of two amino groups at the N- and C-terminals. The selective derivatization of one of those amino groups can fix the protonation site.



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The guanidination by O-methylisourea can selectively derivatize the side-chain of Lys residues, and the resulting guanidyl group has a higher proton affinity than the amino group.36 In fact, guanidination of a Lys-terminated peptide has been demonstrated to increase the yield of protonated peptides in MALDI.36 Because the guanidyl group is the most efficient site of protonation in the peptide, a proton would be localized at the guanidyl group. Therefore, it is expected that HAD-MS/MS analysis of guanidinated model peptides selectively produced C-terminal side fragment ions. Figure 2 shows the HAD-MS/MS mass spectra of the guanidinated model peptides, [T43*+H]+, [T43p*+H]+, and [T43pp*+H]+. As expected, selective observation of the z' series ions, achieved by guanidination of the Lys residue, facilitated the interpretation of the HAD-MS/MS mass spectra. Regarding the determination of the phosphorylated residue, the mass difference of 181 Da between z'6 and z'7 in the HAD-MS/MS spectra of T43p could be assigned as the phosphorylated Thr residue at position 6 (Figure 2b). As for the case of T43p, the sites of phosphorylation in T43pp were determined as the Ser residues at positions 8 and 10 by a series of z' fragment ions (Figure 2c).


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Figure 2. HAD-MS/MS mass spectra of guanidinated peptides, (a) [T43*+H]+, (b) [T43p*+H]+, and (c) [T43pp*+H]+. Asterisks indicate precursor ions.

Next, we analyzed the other model tryptic phosphopeptides, T18p and T19p, by HAD-MS/MS. As was the case in figures 1 and 2, HAD-MS/MS of protonated T18p and T19p showed complex mass spectra (data not shown), whereas the use of Lys guanidination preferentially gave C-terminal side fragments (Figure 3). The HAD-MS/MS spectra clearly indicated that Tyr5 in T18p and Ser6 in T19p were the sites of phosphorylation. It should be noted that the model phosphopeptides, T18p, T19p, T43p, and T43pp were also analyzed by ESI-based ETD-MS/MS.23 Because ETD involves charge reduction of the precursor ion, only multiply-charged precursors can be analyzed by ETD-MS/MS. The ion/ion reaction efficiency in the ETD process is known to depend on the charge state of the precursor and the employment of peptide ions with higher charge states as the precursors for ETD, to dramatically increase the yield of



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fragment ions.16-18 With regard to the tryptic peptide, protonation preferentially occurred at the N-terminal amino group and/or the C-terminal basic residue, and thereby, singly and doubly protonated peptides were mainly generated by ESI. However, double protonation is not enough for the ion/electron reaction of ETD to yield better sequence coverage.40-42 In fact, ETD-MS/MS of doubly protonated phosphopeptides, T18p, T19p, T43p, and T43pp, produced only several fragment ions and did not provide enough useful information for the sequencing, owing to low fragmentation efficiency.42 Therefore, the method of increasing the charge state of the phosphopeptide is necessary to promote the wider utility of ETD for phosphoproteomic analysis, and the use of a zinc complex that selectively bound the phosphate group improved the quality of the ETD-MS/MS spectra of the phosphopeptides.42 Compared with ETD-MS/MS, HAD-MS/MS involved the reaction between the precursor ion and the hydrogen atom. Because the charge state of precursor was maintained during the fragmentation process, all the ions could be used as precursors for HAD-MS/MS. As shown in figures 1–3, HAD-MS/MS provided enough sequence coverage, even when singly protonated phosphopeptides were used as the precursor ions. In regard to the sequencing of the tryptic peptides, HAD showed greater efficiency than ETD. Additionally, HAD-MS/MS would be suitable for de novo sequencing of small peptides, which are often detected as only their singly-charged forms by ESI.


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Figure 3. HAD-MS/MS mass spectra of guanidinated peptides, (a) [T18p*+H]+, and (b) [T19p*+H]+. Asterisks indicate precursor ions.

Next, we have chosen bovine α-casein as a model phosphorylated protein, because the α-casein tryptic digest has often been used to evaluate the performance of mass spectrometry-based analytical methods. Figure 4a shows the MALDI mass spectrum of an α-casein tryptic digest with guanidination. The m/z values of monoisotopic signals in Figure 4a and their assignment were summarized in Table 2. According to the protein digest database, the ions at m/z 1508.64, 1660.81, and 1969.71 were assigned as the phosphopeptides. In order to confirm these ions as phosphopeptides, we first performed the CID-MS/MS analysis. The CID-MS/MS spectra of the ions at m/z 1508.64, 1660.81, and 1969.71 are shown in figure 4b–d. CID of those ions led to the dominant neutral loss of phosphoric acid (H3PO4, 98 Da). The presence of the signal because of the loss of 98 Da provided valuable confirmatory evidence of phosphorylation. Although phosphoric acid loss was observed from the ions at m/z 1508.64 and 1660.81, CID-MS/MS of the ion at m/z 1969.71 resulted in the loss of two phosphoric acids, indicating that two phosphorylation sites were present. As in the case of H3PO4 loss, the CID-MS/MS spectra of the phosphopeptides also showed y' series ions. However, the labile phosphate group was completely removed from the peptides by



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CID, and thereby, all y' ions were observed as non-phosphorylated fragments. Therefore, the location of the phosphorylation sites could not be determined, because of the extensive phosphate loss.

Figure 4. (a) MALDI mass spectrum of guanidinated α-casein tryptic digest. (b-d) CID-MS/MS spectra of the ions at m/z (b) 1508.64, (c) 1660.81, and (d) 1969.71. Asterisks indicate precursor ions.

Table 2. The assignment of ions observed in MALDI mass spectrum of guanidinated α-casein tryptic digest (Figure 4a). The monoisotopic mass of protonated peptides is described as the observed and calculated m/z values.


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Peptide

Sequence

Composition

Observed m/z

Calculated m/z

S2:161-165*

LNFLK*

C32H54N4O7

676.43

676.414

S2:200-205

VIPYVR

C36H60N9O8

746.46

746.456

S2:25-32*

NMAINPSK*

C37H66N13O12S

916.48

916.466

S2:174-181*

FALPQYLK*

C50H77N12O11

1021.59

1021.582

S2:115-125

NAVPITPTLNR

C52H91N16O16

1195.69

1195.680

S1:91-100

YLGYLEQLLR

C60H95N14O16

1267.71

1267.705

S2:81-91*

ALNEINQFYQK*

C63H97N18O19

1409.72

1409.716

S2:138-149*

TVDMEpSTEVFTK*

C60H99N15O26SP

1508.64

1508.633

S1:106-119

VPQLEIVPNpSAEER

C68H115N19O27P

1660.81

1660.794

S1:8-22

HQGLPQEVLNENLLR

C76H127N24O24

1759.95

1759.945

S1:43-58

DIGpSEpSTEDQAMEDIK*

C71H119N20O39P2S

1969.71

1969.712

Next, we analyzed guanidinated phosphopeptides in the α-casein tryptic digest, in order to confirm the applicability of HAD-MS/MS in sequencing of phosphopeptides in a protein digest. Figure 4 shows the HAD-MS/MS spectra of the ions at m/z 1508.64, 1660.81, and 1969.71, which provided the selective observation of the z' series ions. As shown in Figure 4, phosphorylated residues were easily assigned at Ser6 in S2: 138–149, Ser10 in S1: 106–119, and Ser4 and Ser6 in S1: 43–58. It should be noted that the ion at m/z 1660.81 was an arginine (Arg)-terminated peptide. Because the Arg-terminated tryptic peptide had a guanidine group at the C-terminal, z' ions were preferentially observed in the HAD-MS/MS spectrum, as was the case for the guanidinated Lys-terminated tryptic peptides. For comparison of ESI-based ETD-MS/MS, only one phosphorylation site, Ser5 in S1: 106– 119, was determined by direct analysis of the α-casein tryptic digest, even after using the recently reported zinc complex-aid method.42 Therefore, MALDI-based HAD-MS/MS with guanidination



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was re-confirmed as a better method than ESI-based ETD-MS/MS for phosphopeptide sequencing of a protein digest. However, the MALDI does not directly combine with chromatography and the total time for an experiment of HAD-MS/MS is on the order of seconds due to long reaction time for HAD. Therefore, the present HAD-MS/MS based method is potentially useful for off-line analysis of chromatographically-separated tryptic peptide fractions and identification of isolated protein digest, i.e., peptide mass fingerprinting.

Figure 5. HAD-MS/MS spectra of guanidinated α-casein tryptic digest, ions at m/z (a) 1508.64, (b) 1660.81, and (c) 1969.71. Asterisks indicate precursor ions.

CONCLUSIONS We investigated the applicability of MALDI HAD-MS/MS for the analysis of phosphoprotein tryptic digests. The tryptic peptides are categorized as Arg- and Lys-terminated peptides. Because the proton is strongly bound to the guanidine group of the Arg residue,


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HAD-MS/MS spectra of Arg-terminated tryptic peptides preferentially showed the C-terminal side fragment. In contrast, Lys-terminated tryptic peptides have two amino groups, one at each the N- and C-terminus, and these groups were complementally protonated. Therefore, HAD-MS/MS of the Lys-terminated tryptic peptides provided both N- and C-terminal side fragments, which made the attribution of m/z values to a specific series of consecutive c' and z' ions difficult. The guanidination of the Lys residue of the peptide increased the yield of the protonated peptide in MALDI. The protonation site is fixed at the guanidinated Lys residue, which usually presents at the C-terminus in tryptic peptides. Therefore, HAD-MS/MS of guanidinated tryptic peptides produced protonated C-terminal side fragments, as was the case for the Arg-terminated tryptic peptides. The guanidination of tryptic peptides, followed by MALDI HAD-MS/MS analysis facilitates de novo peptide sequencing, including the determination of the phosphorylation site.

ACKNOWLEDGMENTS Authors acknowledge Dr. Takashi Nishikaze (Shimadzu Corporation) for the technical advice of peptide guanidination. This work was supported by JSPS KAKENHI grant number JP26505016.

REFERENCES 1.

Hunter, T., Cell 2000, 100, 113-127.

2. 3.

Cohen, P., Trends Biochem. Sci. 2000, 25, 596-601. Palumbo, A. M.; Smith, S. A.; Kalcic, C. L.; Dantus, M.; Stemmer, P. M.; Reid, G. E., Mass

Spectrom. Rev. 2011, 30, 600-625. 4.

Boersema, P. J.; Mohammed, S.; Heck, A. J., J. Mass Spectrom. 2009, 44, 861-878.

5. 6.

Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W., J. Am. Chem. Soc. 1998, 120, 3265-3266. Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F., Proc. Natl. Acad. Sci.

U S A 2004, 101, 9528-9533. 7.

Kelleher, N. L., Anal. Chem. 2004, 76, 196A–203A.

8.

Coon, J. J., Anal. Chem. 2009, 81, 3208–3215.

9.

Zhou, H.; Ning, Z.; Starr, A. E.; Abu-Farha, M.; Figeys, D., Anal. Chem. 2012, 84, 720-734.



10.

Page 20 of 21

Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B.

K.; McLafferty, F. W., J. Am. Chem. Soc. 1999, 121, 2857-2862. 11. Sobczyk, M.; Anusiewicz, I.; Berdys-Kochanska, J.; Sawicka, A.; Skurski, P.; Simons, J., J. Phys. Chem. A 2005, 109, 250-258. 12.

Syrstad, E. A.; Tureček, F., J. Am. Soc. Mass Spectrom. 2005, 16, 208-224.

13. Asakawa, D.; Yamashita, A.; Kawai, S.; Takeuchi, T.; Wada, Y., J. Phys. Chem. B 2016, 120, 891-901. 14.

Shi, S. D.-H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W., Anal.

Chem. 2001, 73, 19-22. 15.

Kweon, H. K.; Håkansson, K., Anal. Chem. 2006, 78, 1743-1749.

16. Good, D. M.; Wirtala, M.; McAlister, G. C.; Coon, J. J., Mol. Cell Proteomics 2007, 6, 1942-1951. 17.

Frese, C. K.; Altelaar, A. F.; Hennrich, M. L.; Nolting, D.; Zeller, M.; Griep-Raming, J.;

Heck, A. J.; Mohammed, S., J. Proteome Res. 2011, 10, 2377-2388. 18. 19.

Rožman, M.; Gaskell, S. J., Rapid Commun. Mass Spectrom. 2012, 26, 282-286. Kong, R. P.; Quan, Q.; Hao, Q.; Lai, C. K.; Siu, C. K.; Chu, I. K., J. Am. Soc. Mass

Spectrom. 2012, 23, 2094-2101. 20.

Diedrich, J. K.; Julian, R. R., J. Am. Chem. Soc. 2008, 130, 12212-12213.

21.

Diedrich, J. K.; Julian, R. R., Analytical chemistry 2011, 83, 6818-26.

22.

Tureček, F.; Julian, R. R., Chemical reviews 2013, 113, 6691-6733.

23.

Asakawa, D., Mass Spectrom. Rev. 2016, 35, 535-556.

24.

Takayama, M., J. Am. Soc. Mass Spectrom. 2001, 12, 1044-1049.

25.

Kocher, T.; Engstrom, Å.; Zubarev, R. A., Anal. Chem. 2005, 77, 172-177.

26. Asakawa, D.; Calligaris, D.; Smargiasso, N.; De Pauw, E., J. Phys. Chem. B 2013, 117, 2321-2327. 27.

Asakawa, D.; Smargiasso, N.; De Pauw, E., J. Am. Soc. Mass Spectrom. 2013, 24, 297-300.

28.

Asakawa, D.; Smargiasso, N.; De Pauw, E., Anal. Chem. 2014, 86, 2451-2457.

29. Asakawa, D.; Smargiasso, N.; Quinton, L.; De Pauw, E., J. Am. Soc. Mass Spectrom. 2014, 25, 1040-1048. 30.

Asakawa, D.; Takayama, M., J. Am. Soc. Mass Spectrom. 2011, 22, 1224-1233.

31.

Asakawa, D.; Takayama, M., J. Phys. Chem. B 2012, 116, 4016-4023.

32.

Hanisch, F. G., Anal. Chem. 2011, 83, 4829-4837.

33. Asakawa, D.; Calligaris, D.; Zimmerman, T. A.; De Pauw, E., Anal. Chem. 2013, 85, 7809-7817. 34. 35.

Asakawa, D.; Takayama, M., J. Mass Spectrom. 2012, 47, 180-187. Takahashi, H.; Sekiya, S.; Nishikaze, T.; Kodera, K.; Iwamoto, S.; Wada, M.; Tanaka, K.,

Anal. Chem. 2016, 88, 3810-3816. 36.

Beardsley, R. L.; Reilly, J. P., Anal. Chem. 2002, 74, 1884-1890.


Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37.

Fukuyama, Y.; Takeyama, K.; Kawabata, S.; Iwamoto, S.; Tanaka, K., Rapid Commun. Mass

Spectrom. 2012, 26, 2454-2460. 38.

Zubarev, R. A., Mass Spectrom. Rev. 2003, 22, 57-77.

39.

Paizs, B.; Suhai, S., Mass Spectrom. Rev. 2005, 24, 508-548.

40. Asakawa, D.; Takeuchi, T.; Yamashita, A.; Wada, Y., J. Am. Soc. Mass Spectrom. 2014, 25, 1029-1039. 41.

Asakawa, D.; Wada, Y., J. Phys. Chem. B 2014, 118, 12318-12325.

42.

Asakawa, D.; Osaka, I., Anal. Chem. 2016, 88, 12393-12402.


De Novo Sequencing of Tryptic Phosphopeptides Using Matrix

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