213 nm Ultraviolet Photodissociation on Peptide Anions: Radical-Directed Fragmentation Patterns

  • Mohammad A. Halim
  • Marion Girod
  • Luke MacAleese
  • Jérôme Lemoine
  • Rodolphe Antoine
  • Philippe Dugourd
Research Article

Abstract

Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα–C bond but also stimulates the breaking of N–Cα and C–N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N–Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.

Graphical Abstract

Keywords

Photo-fragmentation Radical anions UVPD Peptide TDDFT 

Introduction

Alternative to collision [1, 2, 3] and electron [4, 5] based techniques, photon-based methods have emerged as new powerful approaches for characterizing peptides, polysaccharides and proteins [6, 7, 8, 9, 10, 11, 12]. Among them, ultraviolet photodissociation (UVPD) leads to intense fragmentation patterns. In this method, protein and peptide cations predominately dissociate to a/x ions and less frequently to c/z and b/y ions. Different wavelengths such as 157, 193, 220, and 280 nm have been implemented in UVPD. Above and at 280 nm, specific fragmentation has been reported following excitation of aromatic residues in peptides or proteins [13]. The number of fragment ions increases as the wavelength decreases from 280 to 213 nm [13, 14].

Another efficient and popular wavelength 193 nm has been implemented in hybrid linear ion trap-Orbitrap mass spectrometer for characterizing different peptide and proteins in positive polarity. Wide-ranging fragmentation yields a/x, b/y, c/z, y-1, v, w, and d ions and thus provides nearly complete sequence coverage. Whole protein characterization has been achieved by this technique implementing direct infusion and/or chromatographic time scale [15, 16]. Along with common fragment ions, Madsen et al. also observed some uncommon fragment ions such as a + 2, c – 1 and z + 1 [17]. This study disclosed that fragmentation patterns varied with the protonation state of the peptide. When protonation takes place at N-terminus, cleavage of Cα–C bond occurred; however, N–Cα cleavage is favored with C-terminus protonation.

Thompson et al. employed vacuum photodissociation at 157 nm on singly protonated peptide ions to elucidate the unusual backbone cleavage [18]. Cui et al. further revealed that basic residues in the C-terminal yields to x, v, and w fragment ions, whereas N-terminal produces a and d fragments ions [19]. Moreover, a + 1 and x + 1 radical ions are identified from the charge localized N- and C-terminals, respectively. Secondary radical elimination of hydrogen atom are detected from a + 1 and x + 1 ions to produce a and x ions, respectively. Satellite ions such as d, v, and w are formed due to part of side chain elimination; b, c, and z fragment ions are also noticed but are less frequent than a and x ions. Hydrogen/deuterium exchange experiments further confirmed that both backbone amide and side-chain β-carbon hydrogens can undergo elimination to yield a and x ions [20]. Implementing time-resolved photodissociation at 157 nm revealed some unusual but stable x + 2 fragment ion compared with less common a + 2 ion [21]. They proposed that addition of one hydrogen to x + 1 and a + 1 radical ions can yield x + 2 and a + 2 ions. Migration and transfer of hydrogen atom to radical ions have also been witnessed in ECD studies [22, 23].

However, most of these experiments were conducted on peptide and protein cations and very few were directed on negative polarity. It is assumed that around 50% of naturally occurring peptides are acidic and prone to yield negative ions. Kjeldsen et al. reported Cα–C backbone fragmentation by EDD (electron detachment dissociation) for peptide and observed more C-terminal species (x ions) than N-terminal fragments (a ions) [24]. Comparison of negative electron transfer dissociation (NETD) and UVPD for peptide anion disclosed that NETD usually produce simple set of a/x ions [25]. In NETD, along with a/x ions various neutral losses are observed from entire or partial side-chain cleavage of amino acids [26]. Activated ion negative electron transfer dissociation (AI-NETD) of doubly charged peptide ions also generates some hydrogen loss from a and x fragment ions [27].

Some previous electron photo-detachment dissociation (EPD) studies were performed with UV lasers on peptides and small proteins in negative polarity [28, 29]. Antoine et al. investigated the electron photo-detachment dissociation of peptides using 262 nm with a linear ion trap [30]. Formation of [M – 2H]-• radical anion from the precursor ion was documented in this experiment; a/x and c/z fragment ions were observed [28]. Comparative studies between EDD and EPD revealed significantly different fragment ion distributions in which EPD fragment ions are typically produced from tryptophan and histidine resides whereas in EDD backbone dissociation are favored [28]. However, EDD on small proteins including ubiquitin and melittin suggests that basic resides may promote the formation of a/x fragment ions [31].

Radical-containing peptides promote characteristic fragmentation pattern in mass spectrometry [32, 33]. Radical peptides are classified into two categories: hydrogen-deficient and hydrogen-rich radicals [34]. The former type is typically formed in UVPD, EDD, and NETD routes, whereas the latter is generated from ECD/ETD [8, 24, 35, 36, 37]. Recently, formation of hydrogen-deficient species from the hydrogen-rich radical cation in ECD received great attention because of extensive fragmentation and widespread side-chain loss [33, 38]. Radical migration in hydrogen-deficient peptide radical promotes extensive neutral loss and allows remote backbone dissociation [33, 39].

Here, we present the implementation of 213 nm UVPD in a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. We observed distinctive Cα–C, N–Cα, and C–N backbone fragmentations from the hydrogen-deficient radical anions. Radical-driven extensive neutral loss is likewise evident in these experiments. Moreover, series of hydrogen-deficient and hydrogen-rich fragments are observed.

Material and Methods

Photodissociation Mass Spectrometry

All experiments were performed on a hybrid quadrupole-Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a HESI ion source. Three small peptides YTIAALLSPYS, DYKDDDDK, and RGDSPASSKP were used without any further purification. Peptide samples were prepared at 1 μM concentration in 50/49/1 (v/v/v) acetonitrile/water/ammonium hydroxide and directly infused to MS at a flow rate of 5 μL/min. All spectra were acquired using a mass range of 100–1500 m/z and resolving power of 140,000 at m/z 400. The automatic gain control (AGC) target for MS/MS was set to 1 × 106 and the maximum injection time was set at 250 ms. The isolation width was 2 Th. When required, the identification of fragment ions was confirmed by fragmentation of a single isotope (selection width 0.4 Th). The high collision dissociation (HCD) collision energy was set to the minimum 2 eV in order to avoid collisions and provide photofragmentation spectra free of CID contamination. Different HCD trapping times including 100, 500, 1000, and 2000 ms (2, 10, 20, 40 laser shots, respectively) were considered. All experiments were performed on five microscans mode with averaging 200 scans.

For UVPD experiments, BrillantB Nd:YAG (Quantel, Les Ulis, France) laser was employed. Details of the setup are given elsewhere [14]. In brief, the 5th harmonic (λ = 213 nm) with a repetition rate of 20 Hz was used. The hybrid quadrupole-Orbitrap Q-Exactive mass spectrometer was modified to permit the laser irradiation of peptide ions. The laser beam passes through lenses, diaphragms, and then is introduced in the HCD cell using two dichroic mirrors. A UV grade fused-silica window was fitted on the back of the HCD cell to allow penetration of a laser beam. The laser beam energy irradiating the ions was ~1 mJ/pulse. The laser was slightly off-axis so as to avoid photofragmentation in the C-trap.

Manual analysis of UVPD data was performed with the aid of ChemCalc software [40]. Peak lists of three peptides were generated for all six major UVPD ion types (a, b, c, x, y, and z). Fragments mass tolerance was set to 20 ppm.

Computation

All calculations were conducted with the Gaussian 09 software package [41]. Optimization and subsequent vibrational frequency calculation on the model amide system CH3CONHCH3 were performed using density functional theory employing Becker’s (B3) [42] exchange functional combining Lee, Yang, and Parr’s (LYP) [43] correlation functional. Gaussian basis set 6-311+G (2d,p) was considered. Natural bond orbital (NBO) [44, 45] calculations were computed at the same level of theory. For calculating the excited state properties, time-dependent density functional theory (TDDFT) [46] was employed with the B3LYP/6-311+G(2d,p) level of theory in gas phase. For TDDFT calculation, 20 excited states were considered.

Result and Discussion

The Photodissociation of Peptide 1 (YTIAALLSPYS)

The photodissociation spectrum of the doubly deprotonated [M – 2H]2– (m/z 597.8057) of this peptide is presented in Figure 1a. Exact masses and assignments of fragment ions of this peptide are summarized in Table 1. Similar to previous studies, the characteristic [M – 2H]–• charge-reduced radical species is detected at m/z 1195.6094 Da. This radical species is typically generated from photo-induced electron detachment from the selected peptide precursor. Intense neutral losses are detected from this radical species (Table 2 and Figure 2). Similar neutral losses are also demonstrated in previous studies [30, 31, 47, 48, 49]. The CH3 radical (15.0242 Da) loss appears at m/z 1180.5852 from the side chain of Ala [50]. Neutral losses of CO (27.9947 Da) and CH3CH2 (28.9995 Da) are noticed at m/z 1167.6147 and 1166.6099 Da, respectively. Removal of CH3CH2 can be used to distinguish the side chain loss of Ile (28.9995 Da) or Leu (43.0542 Da) [51]. Loss of CH2O (30.0100 Da) and CH2OH (31.0178 Da) are also observed from the side chain of Ser. NETD study on Ser-containing peptide witnessed the loss of CH2O when Ser is not phosphorylated [26]. The peak at m/z 1151.5829 can be assigned to the loss of C2H4O (44.0265 Da) from Thr side chain [26, 50]. The sequential loss (61.9998 Da) of CO2 and H2O is also identified at m/z 1133.6099. Radical elimination of a C3H8ON from the Thr residue may lead to the fragment ion detected at m/z 1121.5759. Loss of tyrosylate groups from the side chain of Tyr (107.0472 and 106.0406 Da) is identified at m/z 1088.5622 and 1089.5688 Da, respectively. The phenoxy group of the tyrosylate produces an oxygen radical, which induces the cleavage of Cα–Cβ side chain of the tyrosine residue and promotes the formation of O=C6H4=CH2 (exact mass 106.0413 Da) ion [8, 50, 52]. Two relatively weak peaks at m/z 1139.5855 and 1123.5910 can be assigned for the side chain and related ion loss (56.0239 and 72.0184 Da) from Leu or Ile [26, 51, 52, 53]. Combined losses of tyrosylate and C2H4O from Tyr and Thr appear at m/z 1045.5419 and 1044.5346, respectively.
Figure 1

Photodissociation spectra of the doubly deprotonated [M – 2H]2– ion (m/z = 597.8057) of YTIAALLSPYS at 213 nm. The precursor ion is indicated by the * symbol and the neutral losses are indicated by ion masses. The green and blue lines represent the a, b, c and x, y, z ions, respectively. (a) Spectrum of 100–1200 m/z, (b) spectrum of 900–1100 m/z, and (c) spectrum of 600–900 m/z

Table 1

Exact Masses and Assignments of Ions from Backbone Dissociation Detected in the UVPD of Doubly Deprotonated (m/z = 597.8057) of YTIAALLSPYS [M – 2H]2–

Experimental m/z

Theoretical m/z

Assignment

Chemical composition

Mass difference (ppm)

267.0977

267.0981

(y2)

C12H15N2O5

–0.1721

294.0848

294.0852

(x2 + 1).

C13H14N2O6

–0.1477

295.0924

295.0852

(x2 + 2)

C13H15N2O6

2.9264

362.1348

362.1352

(y3 – 2)-

C17H20N3O6

–0.1696

364.1505

364.1509

(y3)

C17H22N3O6

–0.1575

491.2612

491.2744

(a5 + 1).

C24H37N5O6

–5.3107

591.2533

591.2540

(x5 + 1).

C27H37N5O10

–0.2914

604.3579

604.3586

(a6 + 1).

C30H48N6O7

–0.2089

676.3437

676.3432

(y6 – 1).

C32H48N6O10

0.1889

677.3503

677.3510

(y6)

C32H49N6O10

–0.2864

703.3294

703.3303

(x6)

C33H47N6O11

–0.3636

704.3384

704.3381

(x6 + 1).

C33H48N6O11

0.1069

717.4287

717.4425

(a7 + 1).

C36H59N7O8

–5.5519

747.3786

747.3803

(y7 – 1).

C35H54N7O11

–0.6880

748.3876

748.3881

(y7)

C35H55N7O11

–0.2259

759.4406

759.4405

(c7 – 2)

C37H59N8O9

0.0404

761.4556

761.4561

(c7)

C37H61N8O9

–0.2059

773.3587

773.3596

(x7 – 1).

C36H51N7O12

–0.3591

774.3659

774.3674

(x7)

C36H52N7O12

–0.5911

775.3733

775.3752

(x7 + 1).

C36H53N7O12

–0.7867

803.4658

803.4667

(a8)

C39H63N8O10

–0.3571

804.4737

804.4745

(a8 + 1).

C39H64N8O10

–0.3470

805.4815

805.4824

(a8 + 2)

C39H65N8O10

–0.3612

817.4079

817.4096

(y8 – 2)

C38H57N8O12

–0.6472

818.4158

818.4174

(y8 – 1).

C38H58N8O12

–0.6331

819.4256

819.4252

(y8)

C38H59N8O12

0.1435

833.4757

833.4773

(b8 + 2)

C40H65N8O11

–0.6535

845.4032

845.4045

(x8)

C39H57N8O13

–0.5119

846.4109

846.4123

(x8 + 1).

C39H58N8O13

–0.5866

847.4204

847.4201

(x8 + 2)

C39H59N8O13

0.1174

900.5049

900.5195

(a9)

C44H70N9O11

–5.8453

901.5120

901.5273

(a9 + 1).

C44H71N9O11

–6.1741

930.4916

930.4937

(y9 – 2)

C44H69N9O13

–0.8223

944.5335

944.5331

(c9 – 1).

C45H72N10O12

0.1744

945.5378

945.5409

(c9)

C45H73N10O12

–1.2759

957.4795

957.4807

(x9 – 1).

C45H67N9O14

–0.4833

958.4859

958.4886

(x9)

C45H68N9O14

–1.0864

1032.5470

1032.5492

(y10 – 1)

C48H76N10O15

–0.8721

1059.5334

1059.5440

(x10)

C49H75N10O16

–4.2870

1060.5395

1060.5440

(x10 + 1).

C49H76N10O16

–1.8463

1106.5866

1106.5886

(c10 – 2)

C54H80N11O14

–0.8114

1107.5938

1107.5964

(c10 – 1).

C54H81N11O14

–1.0757

Table 2

Exact Masses and Assignments of Neutral Loss Detected in the UVPD of Doubly Deprotonated (m/z = 597.8057) of YTIAALLSPYS [M – 2H]2–

Experimental m/z

Theoretical m/z

Assignment

Chemical composition

Mass difference (ppm)

1195.6094

1195.6119

[M – 2H]–•

C57 H85 N11 O17

–1.0065

1180.5852

1180.5885

[M – 2H-CH3]

C56 H82 N11 O17

–1.3313

1167.6147

1167.6170

[M – 2H – CO]–•

C56 H85 N11 O16

–0.9306

1166.6099

1166.5728

[M – 2H – CH3CH2]

C55 H80 N11 O17

14.9551

1165.6009

1165.6047

[M – 2H – CH2O]–•

C56 H83 N11 O16

–1.5532

1164.5942

1164.5935

[M – 2H – CH2OH]

C56 H82 N11 O16

0.2703

1151.5829

1151.6221

[M – 2H – C2H4O]–•

C55 H81 N11 O16

–15.7951

1150.5783

1150.6143

[M – 2H – COOH]

C56 H84 N11 O15

–14.5355

1139.5855

1139.5504

[M – 2H – C4H8]–•

C53 H78 N11 O17

14.1289

1133.6099

1133.6116

[M – 2H – (CO2+H2O)]–•

C56 H83 N11 O14

–0.6445

1123.5910

1123.5312

[M – 2H – C4H11N]–•

C53 H75 N10 O17

24.1551

1121.5759

1121.5513

[M – 2H – (C3H8ON)]

C54 H77 N10O16

9.9354

1089.5688

1089.5712

[M – 2H – (OC6OH4=CH2)]–•

C50 H79 N11 O16

–0.9454

1088.5622

1088.5633

[M – 2H – (HOC6H4=CH2)]

C51 H78 N11 O16

–0.4512

1045.5419

1045.5449

[M – 2H – (OC6OH4=CH2+C2H4O)]–•

C48 H75 N11 O15

–1.2420

1044.5346

1044.5371

[M – 2H – (HOC6H4=CH2+C2H4O)]–•

C48 H74 N11 O15

–1.0060

987.5129

987.5271

[z10 – CHO]–•

C47H73N9O14

–5.7445

986.5053

986.5193

[z10 – 1 – CHO]

C47H72N9O14

–5.6699

871.5031

871.5162

[a9 – CH3CH2]– •

C43H69N9O10

–5.2771

856.4917

856.5291

[a9 – C2H4O]

C43H70N9O9

–15.0806

789.4493

789.4869

[b8 + 2 – C2H4O]

C39H65N8O9

–15.1617

597.8057

597.8059

[M – 2H]2–

C57 H85 N11 O17

–0.1139

575.7925

575.8111

[M – 2H – CO2]2–

C56 H85 N11 O15

–7.4861

205.0700

205.0972

[y2 – CO2 + H2O]

C11H13N2O2

–10.9548

Figure 2

Side-chain losses detected from peptide 1, YTIAALLSPYS at 213 nm

Zooms of Figure 1a are shown in Figure 1b, c, and Supplementary Figure S1. Selected fragment ions from the single isotope selection of the doubly deprotonated [M – 2H]2– precursor ions are shown in Supplementary Figure S2. For peptide 1, a series of radical (an + 1). fragment ions is observed for n = 5, 6, 7, 8, and 9 These ions correspond to the elemental composition of an ions plus one hydrogen atom (explaining the +1 in the notation) and are radicals (dot in the notation). This nomenclature is in agreement with the one proposed recently by Chu et al [54] except that we do not include the hydrogen symbol (H) after the number of losses or gains. Homolytic cleavage between the Cα and the carbonyl C from the precursor ion induced the formation of these radical ions, as shown in Scheme 1. Classic (an) fragment ions are detected for n = 8 and 9. These ions may mainly arise from the fragmentation of the doubly deprotonated [M – 2H]2– precursor ion. However, they can also be produced by secondary H elimination from the radical (an + 1). fragment ions [19]. Abundant a ions are favored by aromatic amino acids and in this case it is due to Tyr residue in N-terminal [28, 52]. An unusual fragment such as (a8 + 2) is additionally identified at m/z 805.4815, which may be due to the presence of Pro residue [14, 17]. Detection of (a + 2) is also reported by Madsen et al. in a high-throughput UVPD study in negative polarity for complex proteomic sample [55]. Two peaks at m/z 871.5031 and 856.4917 correspond to the loss of CH3CH2 (28.9995 Da from Ile) and C2H4O (44.0265 Da from Thr) from (a9) ion. Radical (xn + 1). ions are also formed via homolytic cleavage of the Cα–carbonyl C bond, complementary to (an + 1). ions (Scheme 1). Series of radical (xn + 1). ions are noticed at n = 2, 5, 6, 7, 8, and 10, whereas (xn) ions are detected at n = 6, 7, 8, and 9. Two unusual fragment types such as (xn + 2) for n = 2, 8, and radical (xn – 1). for n = 7 and 9 appear for peptide 1, and (x2 + 2) ion detected at m/z 295.0924 is close to Pro residue [14]. Kim and Reilly found xn + 2 fragment ions at 157 nm UVPD and concluded that some x + 1 radical ions may take one hydrogen to form these new ions [21]. (xn + 2) ions are also detected at 193 nm UVPD [55]. The proposed fragmentation pathway for the formation of (x2 + 2) ion is presented in Scheme 2.The formation of two (xn – 1). ions are likewise attributable to the radical elimination of hydrogen atom from the corresponding xn ions. Shaw et al. also observed some (xn – 1). ions in activated ion negative electron transfer dissociation [27]. Moreover, classic fragmentation of the Cα–C bond with proton transfers from the charge-reduced [M – 2H]–. radical species also yields to the formation of (xn – 1). ions. Indeed, these ions will contain the initial radical site and the negative charge. Fragmentation is then observed after electron photo-detachment.
Scheme 1

Proposed mechanism for the formation of (an + 1). and (xn + 1). fragment ions during UVPD of doubly deprotonated peptide [M – 2H]2–

Scheme 2

Proposed mechanism for the formation of (x2 + 2) product ion from the (x1 + 1). fragment ions during the UVPD of the doubly deprotonated YTIAALLSPYS peptide

Series of (yn) ions are detected at n = 2, 3, 6, 7, and 8. Radical (yn – 1). ions are also observed at the positions n = 6, 7, 8, and 10. These ions arise from the homolytic cleavage of the C–N bond from the precursor ion (Scheme 3). However, complementary (bn + 1). radical ions are not detected. Fragmentation of the C–N bond from the charge-reduced [M – 2H]–. radical species may also leads to the formation of the (yn – 1). ions, if the charge and the radical site after electron loss are located on the C-terminal side. As a general statement, the abundance of fragment ions results from both direct fragmentation of the precursor ions and fragmentation of the charge-reduced radical ions obtained after electron loss (EPD). (yn – 1). radical ions could also be formed by H elimination from the (yn) ions. Three new (yn – 2) ions are detected for this peptide at n = 3, 8, and 9 positions and could be formed by H elimination from the (yn – 1). ions. The fragmentation of the C–N bond close to the Pro residue can also explain the formation of the (y3 – 2) fragment ion [14]. Once again, these fragment ions could also arise from the homolytic cleavage of C–N bond fragmentation from the charge-reduced [M – 2H]–. radical species. One (b8 + 2)- fragment ion is detected at m/z 833.4757 for this peptide attributable to the presence of the Pro residue [14]. A neutral loss of 44.0264 Da corresponds to C2H4O of Thr observed at m/z 789.4493 from (b8 + 2)- (Figure 1a).
Scheme 3

Proposed mechanism for the formation of (bn + 1). and (yn – 1). fragment ions during UVPD of doubly deprotonated peptide [M – 2H]2–

c/z Ions are less abundant for this peptide. Two (cn)- ions are detected at n = 7 and 9 positions. Moreover, two (cn – 1). ions at n = 9, 10 positions and (cn – 2) ions at n = 7, 10 sites are observed. Radical (cn – 1). ions could be produced via the homolytic cleavage of the N–Cα bond from the precursor ion (Scheme 4). Hydrogen abstraction from c ions are also detected in ECD [22, 56, 57]. The formation of the (cn – 2) ions could be explained by the radical induced fragmentation of the N–Cα bond from the charge-reduced [M – 2H]–. radical species after electron loss.
Scheme 4

Proposed mechanism for the formation of (cn – 1). and (zn + 1). fragment ions during UVPD of doubly deprotonated peptide [M – 2H]2–

The Photodissociation of Peptide 2 (DYKDDDDK)

The photodissociation spectrum of the doubly deprotonated [M – 2H]2– (m/z 505.1906) of peptide DYKDDDDK is presented in Figure 3 and Supplementary Figure S3. Exact masses and assignments of fragment ions of this peptide are summarized in Table 3. Intense neutral losses are also evident from this peptide (Supplementary Table S1). Loss of H2O from the charge-reduced radical species [M – 2H]–• is detected at m/z 992.3709. Losses of one, two, and three CO2 are identified at m/z 966.3913, 922.4019, and 878.4116, respectively. Madsen et al. observed one and two CO2 loss at 193 nm UVPD of singly and multiply charged peptide anions [49]. Abundant CO2 loss was moreover demonstrated in electron detachment dissociation for peptide and protein [29, 30]. Elimination of several CO2 is a common feature related to aspartic and glutamic acid residues in NETD, Al-NETD, EDD, and UVPD [27, 30]. The UVPD spectrum showed losses of 27.9955 Da from [M – 2H]–• that can be attributed to CO, similar to peptide 1. Loss of CO from radical species is also found in an earlier ECD study [58]. The peaks at m/z 903.3321 and 904.3394 correspond to the losses of tyrosylate groups of Tyr (107.0491 and 106.0418 Da) from the [M – 2H]–•. Radical C3H6O2N (88.0371 Da) group elimination from the aspartic amino acid yields to the ion detected at m/z 922.3441. The ion observed at m/z 938.3961 can be assigned to the loss of C3H4O2 (71.9851 Da) from Asp residue [26]. Loss of Lys residue (100.0736 Da) is also detected at m/z 910.3076. Moreover, a loss of 71.0713 Da (C4H9N) observed for the ion at m/z 939.3099 is from the Lys residue [26]. A combined loss of CO2 and H2O appears at m/z 948.3803.
Figure 3

Photodissociation spectra of the doubly deprotonated [M – 2H]2- ion (m/z = 505.1906) of DYKDDDDK at 213 nm (the precursor ion is indicated by the * symbol and neutral losses are indicated by ion masses). The green and blue lines represent the a, b, c and x, y, z ions, respectively

Table 3

Exact Masses and Assignments of Ions from Backbone Dissociation Detected in the UVPD of Doubly Deprotonated (m/z = 505.1906) of DYKDDDDK [M – 2H]2–

Experimental m/z

Theoretical m/z

Assignment

Chemical composition

Mass difference (ppm)

113.0339

113.0113

(b1 – 1).

C4H3NO3

9.1566

114.0179

114.0191

(b1)

C4H4NO3

–0.4551

242.1134

242.0903

(z2 – 1).

C10H14N2O5

9.3508

243.0975

243.0981

(z2)

C10H15N2O5

–0.2527

249.0869

249.0875

(a2)

C12H13N2O4

–0.2630

251.0926

251.1032

(a2 + 2)

C12H14N2O4

–4.2771

286.1034

286.1039

(x2)

C11H16N3O6

–0.2139

288.1197

288.1196

(x2 + 2)

C11H18N3O6

0.0724

357.1405

357.1172

(z3 – 1).

C14H19N3O8

9.3899

358.1252

358.1250

(z3)

C14H18N3O8

0.0647

374.1324

374.1438

(y3 – 1).

C14H22N4O8

–4.5965

375.1509

375.1516

(y3)

C14H23N4O8

–0.2818

376.1976

376.1747

(a3 – 1).

C18H24N4O5

9.2708

377.1818

377.1825

(a3)

C18H25N4O5

–0.2723

401.1300

401.1309

(x3)

C15H21N4O9

–0.3442

402.1380

402.1387

(x3 + 1).

C15H22N4O9

–0.2656

404.1926

404.1696

(b3 – 1).

C19H24N4O6

9.2932

471.1592

471.1363

(z4 – 2)

C18H23N4O11

9.2294

472.1673

472.1442

(z4 – 1).

C18H24N4O11

9.3202

490.1778

490.1785

(y4)

C18H28N5O11

–0.3057

491.1933

491.1864

(y4 + 1).

C18H29N5O11

2.7890

492.1965

492.2094

(a4)

C22H30N5O8

–5.2316

493.2165

493.2173

(a4 + 1).

C22H31N5O8

–0.3199

516.1568

516.1578

(x4)

C19H26N5O12

–0.3898

519.2194

519.2043

(b4 – 1).

C23H29N5O9

6.0706

536.2224

536.2231

(c4 – 1).

C23H32N6O9

–0.2609

537.2301

537.2309

(c4)

C23H33N6O9

–0.3274

587.1941

587.1711

(z5 – 1).

C22H30N5O14

9.2746

605.2049

605.2055

(y5)

C22H33N6O14

–0.2439

606.2282

606.2287

(a5 – 1).

C26H34N6O11

–0.1597

607.2356

607.2364

(a5)

C26H35N6O11

–0.3272

608.2435

608.2442

(a5 + 1).

C26H36N6O11

–0.2848

631.1839

631.1847

(x5)

C23H31N6O15

–0.3064

634.2464

634.2235

(b5 – 1).

C27H34N6O12

9.2706

651.2492

651.2500

(c5 – 1).

C27H37N7O12

–0.3387

652.2569

652.2578

(c5)

C27H38N7O12

–0.3569

715.2885

715.2661

(z6 – 1).

C28H41N7O15

9.0636

716.2965

716.2739

(z6)

C28H42N7O15

9.1301

721.2544

721.2555

(a6 – 1).

C30H39N7O14

–0.4393

722.2624

722.2633

(a6)

C30H40N7O14

–0.3728

723.2722

723.2711

(a6 + 1).

C30H41N7O14

0.4320

732.2915

732.2926

(y6 – 1).

C28H44N8O15

–0.4529

733.29893

733.3004

(y6)

C28H45N8O15

–0.6083

749.2729

749.2504

(b6 – 1).

C31H39N7O15

9.0999

759.2785

759.2797

(x6)

C29H43N8O16

–0.4891

760.2859

760.2875

(x6 + 1).

C29H44N8O16

–0.6767

761.2953

761.2953

(x6 + 2)

C29H45N8O16

–0.0009

766.2783

766.2769

(c6 – 1).

C31H42N8O15

0.5476

767.2834

767.2848

(c6)

C31H43N8O15

–0.5397

837.2890

837.2903

(a7)

C34H45N8O17

–0.4950

838.2963

838.2980

(a7 + 1).

C34H46N8O17

–0.7068

864.2765

864.2774

(b7 – 1).

C35H44N8O18

–0.3536

865.2866

865.2852

(b7)

C35H45N8O18

0.5561

878.3293

878.3294

(z7-1).

C37H50N8O17

–0.0574

879.3587

879.3372

(z7)

C37H51N8O17

8.6466

881.3027

881.3039

(c7 – 1).

C35H49N9O18

–0.4783

882.3102

882.3117

(c7)

C35H48N9O18

–0.6175

894.3471

894.3481

(y7 – 2)

C37H54N9O17

–0.4140

895.3538

895.3559

(y7 – 1)

C37H53N9O17

–0.8800

921.3364

921.3352

(x7 – 1).

C38H51N9O18

0.4656

922.3421

922.3430

(x7)

C38H52N9O18

–0.3594

A complete series of (an)- fragment ion is observed for this peptide for n = 2–7; (an + 1). ions are detected for n = 4, 5, 6, and 7. These ions are formed via homolytic cleavage from the precursor ion (Scheme 1). Radical (an – 1). ions are detected for n = 3, 5, and 6. Fragmentation of the Cα–C bond from the charge-reduced radical species [M – 2H]–• is involved to produce these series. Secondary radical elimination of hydrogen atom from (an) ions could also yield to the formation of these ions. A complete series of (xn)- fragment ions is detected at n = 2–7 similar to complementary (an) ions. Two radical (xn + 1). ions (n = 3 and 6) are detected at m/z 402.1380 and 760.2859, respectively. Moreover, two (xn + 2) ions (n = 2 and 6), which are formed by addition of one extra hydrogen atom to (xn + 1) ions are detected. Additionally, (x7 – 1) ion is observed at m/z 921.3364. Same fragmentation mechanisms are proposed for the formation of these ions than for the peptide 1 described previously. A distinctive peaks at m/z 886.3281 corresponds to the loss of two H2O molecules from (x7), respectively.

Two (bn) fragment ions are observed at n =1 and 7 sites, whereas very abundant radical (bn – 1). ions are detected for n = 1, 3–7. These ions would come from the fragmentation of the C–N bond from the charge-reduced [M – 2H]–• radical species. Several (yn) ions appear at n = 3–6 positions. Some (yn – 1). ions at n = 3, 6, 7 sites are also detected (formed via the mechanism proposed Scheme 3) as well as (y7 – 2) ion. Specific radical induced fragmentation of the [M – 2H]–• radical species is then also observed, after electron loss, for this peptide.

Cleavage of N–Cα bonds produces series of c and z ions. Four (cn) ions and (cn – 1). radical ions are noticed at n = 4–7 positions. These ions arise from the homolytic cleavage of the N–Cα bond from the precursor ion (Scheme 4). However, complementary (zn + 1) radical ions are not detected. (zn) ions are detected from 2, 3, 6, and 7 positions. Interestingly, complete series of radical (zn – 1). ions (n = 2–7) is observed for this peptide. Classic fragmentation of the N–Cα bond with proton transfers from the [M – 2H]–• radical species is proposed for the formation of these ions as well as the (cn – 1). series. Compared with the first peptide, abundance of c and z ions is noticeable for this peptide and may be due to the presence of five Asp residues. Removal of one H2O, one CO2, and combined CO2 and H2O from (z2) ion are detected at m/z 225.0868 199.1074 and 181.0967, respectively. Previous studies also noticed the losses of H2O and CO2 from z ion when peptide contained Asp residues [56]. Combinations of backbone cleavages and neutral losses are listed in Supplementary Table S1.

The Photodissociation of Peptide 3 (RGDSPASSKP)

The photodissociation spectrum of the doubly deprotonated [M – 2H]2– (m/z 499.2393) of peptide RGDSPASSKP is presented in Figure 4 and Supplementary Figure S4. Exact masses and assignments of fragment ions of this peptide are summarized in Supplementary Table S2. Intense neutral losses are summarized in Supplementary Table S3. The loss of H2O from the charge-reduced radical species [M – 2H]–• (m/z 998.4767) is noticed at m/z 980.4673 (Figure 4a). There are three Ser residues in this peptides and loss of CH2O (30.0095 Da) at m/z 968.4672 can be attributed to the side chain of Ser. The loss of 60.0540 Da observed for the peak at m/z 938.4227 corresponds to the C2H6ON group of the Ser residue. Loss of CO2 (exact mass 43.9895 Da) from the carboxyl group located in C-terminal or side chain of aspartic acid appears at m/z 954.4872. Two distinctive peaks at m/z 899.3982 and 912.4072 correspond to the losses of 99.0785 and 86.0695 Da from the arginine side chain [26, 53]. Loss of 88.0498 Da, which is detected at m/z 910.4269, is related to the side chain of Asp [59].
Figure 4

Photodissociation spectra of the doubly deprotonated [M – 2H]2– ion (m/z = 499.2393) of RGDSPASSKP at 213 nm (the precursor ion is indicated by the * symbol and neutral losses are indicated by ion masses). The green and blue lines represent the a, b, c and x, y, z ions, respectively

Nearly complete series of (an) fragment ions is observed for this peptide for n = 2–9, whereas (an – 1). ions are detected for n = 6 and 9. Radical (an + 1). ions are detected for n = 2–9 (Scheme 1). Addition of one hydrogen to (an + 1). radical ions (similar as shown in Scheme 2 for the xn + 1 ions), which yield (an + 2) is also prevalent for n = 3–5, 7–9 positions; (an + 2) ions are also observed for Proline containing peptides [14, 60] and explain the formation of (a4 + 2). and (a9 + 2) ions. An almost complete series of (xn) fragment ions is detected at n = 1–4, 6–9 similar to the complementary (an) ions. Four (xn – 1). ions are observed for n = 1, 4, 7–9 sites. Moreover, (xn + 1). ions are detected for n = 1–4, 6, 7, and 9. Three (xn + 2) ions (n = 2, 3, 6, and 7) are also formed via H addition on the (xn + 1). ions.

(bn) and (yn) fragments ions are predominant in this peptides, which may be due to the presence of basic Arg and Lys amino acids [61]; (bn) ions are identified for n = 1–5, 8, and 9 positions only missing n = 6 and 7 related to Ala–Ser and Ser–Ser amide bonds; (bn + 1). ions are detected for n = 4, 5, and 9 (Scheme 3). Three (bn – 1). ions are observed at n = 3, 8, and 9. Representative (bn + 2)- ions appear at 2, 4, 9 positions in which two sites (4 and 9) are closed to the Pro residues; (b2 + 2) ion could be explained by the H addition on the (bn + 1). ion. Complete sequence of (yn) ions are found (n = 1-3, 5–9) whereas (yn – 1). ions are noticed for n = 2, 5–9. Distinctive (yn – 2) ions are detected for n =1, 2, 6–9.

Homolytic cleavage and fragmentation, associated with proton transfers, of N–Cα bonds is also noticeable. Full sequence of (cn) ions located for n = 1–3, 5–9, and (cn – 1). ions are noticed at n = 3, 6–9. Fragment (cn – 2) ions are detected for n = 2, 3, 6–8. Similar to peptide 2, complete series of (zn) ions (n =2–9) are generated from this peptide; (zn – 1). ions are also observed for n = 3, 7–9. Moreover, (zn + 1). ions are detected for n =2, 4, 6, 7, and 9 (Scheme 4).

Photo-Induced Hydrogen loss at 213 nm

A general trend is observed for those three peptides with series of backbone cleavages leading to ions deficient in hydrogen. All three peptides produce the distinctive doubly deprotonated [M – 2H]–• charge-reduced radical species upon irradiation of the monoisotopic precursor ion [M – 2H]2–, along with hydrogen loss from the charge-reduced radical species as shown in (Figure 5). Time-dependent density functional theory (TDDFT) calculation has been performed on a model amide system to elucidate the role of πσ* excited state in the photodissociation of peptide. The potential energy surface of the model amide system, π, π*, and σ* molecular orbitals are displayed in Figure 6. The lowest ππ*, πσ*, and electronic group state (S0) are shown with respect to the N–H stretching coordinate of the model amide. The ππ* excitation is observed for the amide system at 215 nm (5.75 eV), which relates with our UVPD experiment at 213 nm. The diffuse and polar character of σ* orbital is observed which is similar to the previous studies on pyrrole/indole system [62, 63]. The shallow barrier with respect to N–H stretch indicates the repulsive nature of this state [62]. For this amide system, the ππ* surface is above the πσ* surface, which may allow the fast internal crossing from the ππ* to the πσ* states and lead to H atom dissociation [63, 64, 65]. The ππ* excitation-induced amide hydrogen loss then provides a general route for the formation of hydrogen-deficient ions in 213 nm UVPD. Repetition of this mechanism with absorption of several photons can lead to fragments displaying multiple H-loss. Moreover, the ππ* excitation-induced amide hydrogen loss may yield a nitrogen-centered amide anion intermediate and stimulate the widespread backbone fragmentation. However, detailed theoretical calculations are sought to elicit the mechanism of radical-driven side-chain loss and backbone fragmentation at 213 nm photodissociation on peptide and protein anions. A similar mechanism can also arise on other bonds from aromatic cycles or COO chromophore groups.
Figure 5

Photodissociation spectra of the doubly deprotonated [M – 2H]2– ion of three peptides. Loss of hydrogen is observed from the characteristic [M – 2H]–• charge-reduced radical at single isotope selection of the doubly deprotonated [M – 2H]2– precursor ions

Figure 6

Potential energy surface of the lowest ππ*, πσ*, and electronic ground state (S0) as a function of the NH stretch reaction coordinate. The optimization, natural bond orbital (NBO), and TD-DFT calculations have been performed at the B3LYP/6-311+G(2d,p) level of theory

Conclusion

The key features of these experiments can be summarized as follows: (1) Extensive sequence specific side-chain losses are observed for all three peptides. (2) Near complete series of classic backbone cleavages (a/x, b/y, c/z) are observed. (3) Unusual fragment ions including (x + 1)., (x + 2), (x – 1)., (y – 1)., (y – 2), (z – 1)., (z + 1)., (z + 2) , and (a – 1)., (a + 1)., (a + 2), (b – 1)., (b + 1)., (b + 2), (c – 1)., (c – 2) are consistently observed in these experiments and further confirmed by selecting single isotopic peak of the precursor ions. Some of these ions are coming from homolytic cleavages of the backbone from the precursor doubly charged ion. Classic fragmentation of backbone bonds concerted with proton transfers and homolytic cleavages are also observed for the charge-reduced [M – 2H]–. radical species after electron photo-detachment. Radical-induced specific fragment ions are then produced in these experiments of UVPD in the negative mode. Some of these ions may also result from secondary H eliminations. (4) Hydrogen-deficient ions may result from ππ* excitation-induced amide hydrogen loss. This ππ* excitation is reached upon absorption of a photon at 213 nm. The present study outlines the difficulty to interpret and systematically analyze the wealth of fragmentation produced by irradiation of peptide and protein anions at the onset of the amide bond absorption band, which may be different from VUV excitation.

Notes

Acknowledgments

The research leading to these results has received funding from the European Research Council under the European Union’s 7th Framework Program (FP7/2007-2013 grant agreement N°320659).

Supplementary material

13361_2015_1297_MOESM1_ESM.docx (2.5 mb)
ESM 1(DOCX 2577 kb)

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Copyright information

© American Society for Mass Spectrometry 2015

Authors and Affiliations

  • Mohammad A. Halim
    • 1
  • Marion Girod
    • 2
  • Luke MacAleese
    • 1
  • Jérôme Lemoine
    • 2
  • Rodolphe Antoine
    • 1
  • Philippe Dugourd
    • 1
  1. 1.Institut Lumière MatièreUniversité Lyon 1 – CNRS, Université de LyonVilleurbanne CedexFrance
  2. 2.Institut des Sciences AnalytiquesUniversité Lyon 1 – CNRS, Université de LyonVilleurbanne CedexFrance

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