Intermolecular condensation products formed during the pyrolysis of peptides

Journal of Analytical and Applied Pyrolysis 92 (2011) 217–223

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Intermolecular condensation products formed during the pyrolysis of peptides Chenglin Liu, Franco Basile ∗ Department of Chemistry, 1000 E. University Ave (Dept. 3838), University of Wyoming, Laramie, WY 82071, United States

a r t i c l e

i n f o

Article history: Received 29 March 2011 Accepted 23 May 2011 Available online 28 June 2011 Keywords: Pyrolysis Thermal degradation Peptide condensation Crosslink Non-volatile products ESI and MALDI–MS

a b s t r a c t Mass Spectrometry (MS) analysis of pyrolysis products of simple peptides has revealed several nonvolatile thermal degradation products at masses lower than the precursor peptide. In addition to these products, many other signals were also observed at higher masses than the precursor peptide, and their characterization is the focus of this study. Here we report on the observation of homo and hetero condensation peptide products formed during the pyrolysis of peptides. The observed peptide condensation products are formed between two, three or even four peptides. Tandem MS (MS/MS) analyses of these products showed that C-terminal to N-terminal intermolecular bonding is preferred during pyrolysis when combining two peptides, rather than involving crosslinking between basic and acidic side chain groups like arginine and aspartic acid. These observations are rationalized by steric hindrance effect and known pKa values of the peptide C- and N-termini and amino acid side groups like aspartic acid and arginine. Pyrolysis of a standard N-acetylated peptide showed no detectable condensation and/or crosslinked products, even in peptides with basic side groups, providing further evidence for the C-terminus to N-terminus intermolecular bonding between peptides under pyrolytic conditions. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The development of ESI–MS [1,2] and MALDI–MS [3] has made possible the analysis of high molecular mass molecules residues from pyrolysis of synthetic polymers [4] and biomolecules [5]. Several investigations have since focused on the nature of the thermal degradation and cleavage reactions induced in small peptides and large proteins. For example, our laboratory showed that aspartic acid containing peptides (Asp, single letter symbol = D), upon pyrolysis at 220–250 ◦ C, undergo site-specific cleavage via hydrolysis at the C-terminus of D when the reaction is carried out under atmospheric conditions [6]. This strategy for protein site-specific (i.e., amino acid specific) “digestion” using pyrolysis also allowed protein identification via database matching of a tandem mass spectrum of a peptide derived from the pyrolysis of an intact protein. Combined, our previous work conclusively determined that protein sequence is conserved even after being subjected to the harsh conditions required for pyrolysis and that protein identification is possible using pyrolysis as the digestion step in a bottom-up proteomic workflow. The pyrolysis of proteins and peptides has been carried out also in simple furnace-type pyrolyzers and on membrane heaters interfaced with DESI–MS and MALDI–MS detection [7,8]. Also, atmospheric pressure thermal dissociation has been

∗ Corresponding author. Tel.: +1 307 766 4376. E-mail address: [email protected] (F. Basile). 0165-2370/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jaap.2011.05.011

reported on peptide and protein ions in the gas phase, showing a more extensive fragmentation pattern (i.e., bn , yn , and an -ions) [9] than the fragmentations observed in condensed phase pyrolysis of peptides and proteins. However, because of the wide range of chemical functionalities present in proteins, the possibility exists for many side reactions to occur during the condense phase pyrolysis of proteins. In fact, many side reactions have been identified and include dehydration [6,10] and deamination [6]. Recently the formation of cyclic peptides associated with dehydration products during pyrolysis was studied and confirmed for the peptide YGGFMRGL using LC–MS and cyclic peptide reference standards [11]. Also, the formation of crosslinked products during the pyrolysis of amino acids has been observed, albeit, in the presence of the methylating reagent tetramethylammonium hydroxide (TMAH) to allow for GC–MS analysis of the products. This study found that most amino acids form N-terminus to C-terminus dimmers followed by cyclization (5 or 6 member rings) involving side chain groups, while cysteine, isoleucine, methionine and valine only generated non-cyclic dimmers [12]. It is expected that the complexity of the formed dimmers would increase if mixtures of two different amino acids are considered. The overall picture emerging from all these studies is that much research remains to be done before pyrolysis can be applied to the analysis of complex biological system usually studied in proteomics. As part of this continuing effort to characterize nonvolatile pyrolysis products of proteins and peptides, we present in this study evidence for the formation of hetero and homo

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intermolecular condensation products between peptides when pyrolyzed at 220 ◦ C in the condensed phase. Using tandem MS (MS/MS) measurements and peptides with unique side groups and/or modifications (e.g., N-acetylated) the preferential formation of non-volatile products with C-terminus to N-terminus bonds between peptides was inferred. 2. Experimental 2.1. Chemicals The peptides (A) Angiotensin II, human, sequence DRVYIHPF; (B) HA peptide, sequence YPYDVPDYA; (C) Leu-enkephalin, sequence YGGFL; and (D) Acetylated Angiontensin II, human, sequence AcDRVYIHPF (all from AnaSpec, San Jose, CA) were used without further purification. Solutions of water and methanol (HPLC grade, Burdick & Jackson, Muskegon, MI) and formic acid (96%, ACS Reagent grade, Aldrich, St. Louis, MO) were used for all sample preparations and ESI–MS measurements. 2.2. Pyrolyzer design and pyrolysis procedure Samples were placed in a glass tube (length 31 mm and internal diameter 4 mm; Agilent, Santa Clara, CA, Part # 5180-0841) and heated using a resistance heating wire (Omega, Stamford, CT, nickel–chromium wire, part # NI60-015-50, length 20 cm) enwound around the tube, powered by 13 V alternating current (AC). Temperature was measured in situ using a thermocouple probe (model HH12A, Omega Company, Stamford, CT) reaching down the bottom of the glass tube. Approximately 1 mg solid sample of the standard peptides was pyrolyzed under ambient conditions. For mixtures of peptides equimolar amounts (about 0.5 mg) of Leu-enkephalin was mixed with HA peptide and also pyrolyzed under ambient conditions. The sample was heated for 10 s under ambient conditions to a final temperature between 180 ◦ C and 300 ◦ C. The nonvolatile pyrolysis residue was collected by washing/extracting the inside of the tube with 1 mL of a 50/50 (v/v) methanol–water solution with 0.1% formic acid (FA). 2.3. Mass Spectrometry The extracted solution of pyrolysis products was directly analyzed using a quadrupole ion-trap MS (LCQ Classic, Finnigan, San Jose, CA) equipped with a nano-Electrospray Ionization (nano-ESI) source by infusing it into the mass spectrometer at a flow rate of 3 ␮L/min using a 250 ␮L syringe. Tandem MS (MS/MS, low energy CID) was conducted with the following parameters: activation q of 0.250; isolation width was 1 amu, and the percentage relative collision energy was in the range of 25–40% and was adjusted such that the relative abundance of the precursor ion in the product ion mass spectrum was approximately 30–50% relative intensity. MALDI–MS measurements were conducted using a MALDI Time-of-Flight MS (Voyager DE-STR, Applied Biosystems, Foster City, CA) instrument equipped with a N2 laser and operated in the reflectron mode. The matrix ␣-cyano-4-hydroxy-cinnamic acid (CHCA) (Aldrich, St. Louis, MO) was used for all measurements and was prepared by dissolving 10 mg of CHCA in a 1 mL solution of 1:1 acetonitrile/water with 0.1% trifluoroacetic acid (TFA) (Pierce Chemical Co., Rockford, IL). The extracted solution of pyrolysis products was directly mixed with the matrix at different volume ratios and air-dried onto a MALDI plate. 3. Results and discussion The MALDI–mass spectrum of the non-volatile pyrolysis (220 ◦ C) products of the peptide Angiotensin II (amino acid

Table 1 Ion signals of detected crosslinked products from the pyrolysis of Angiotensin II peptide. m/z, [M+H]+ ions

Proposed peptide combination* (r = R–NH3 )

1924.4 1942.4 2022.2 2038.9 2056.0 3031.4

RVYIHPF + DrVYIHPF–H2 O RVYIHPF + DrVYIHPF DrVYIHPF + DrVYIHPF–H2 O DrVYIHPF + DrVYIHPF DRVYIHPF + DrVYIHPF DrVYIHPF–H2 O + DrVYIHPF–H2 O + DrVYIHPF–H2 O

*

Also detected, but not shown are permutations of these combinations.

sequence: DRVYIHPF) is shown in Fig. 1b. A cluster of signals near m/z 1000 corresponds to signals for the pyrolysis products due to water loss (m/z 1028), ammonia loss (m/z 1029), water plus ammonia losses (m/z 1011) and aspartic acid cleavage at C-terminal (m/z 931). In addition, a cluster of signals was also observed near m/z 2000 and they correspond to the linear combinations (minus water) between any two of the products detected in the cluster of signals around m/z 1000. Similarly, another signal cluster was observed near m/z 3000, most likely the result of the crosslink between any three of the product detected near m/z 1000. A closer look at the cluster of signals near m/z 2000 reveals that the signal at m/z 1942 corresponds to the crosslink between the peptides RVYIHPF (resulting from the loss of D after pyrolysis) and DrVYIHPF (where r = R–NH3 ). Similarly, for the crosslink of peptides in the signal cluster near m/z 3000, the signal at m/z 3031 corresponds to the crosslink of peptides DRVYIHPF–H2 O, DRVYIHPF–H2 O and DrVYIHPF–H2 O. An extended list of the detected ions and their proposed origin is shown in Table 1. If we assume a C-terminus to N-terminus condensation (vide infra), loss of water occurs during the formation of each new peptide bond between reacting peptides. Additional water losses are induced at the aspartic acid side chain, while ammonia losses occur from the arginine side chain under these experimental conditions. When the pyrolysis temperature was raised to 270 ◦ C, the signals corresponding to the crosslink of two, three and even four peptides were observed and the resulting mass spectrum is shown in Fig. 1c. This temperature dependence of the formation of peptide crosslinked products during pyrolysis was studied in detail for the peptide angiotensin II and results are shown in Fig. 2. The percentage value for the pyrolysis D-cleavage process was calculated as follows: %Cleavage at D =



I(m/z 931) I(crosslinked products) + I(m/z 931) + I(other products)

×100 where the signals for crosslinked products correspond to signals at the clusters at m/z 2000s (2 peptides crosslinked), m/z 3000s (3 peptides crosslinked) and m/z 4000s (4 peptides crosslinked). For the percentage values for the formation of all observed crosslink products, their intensities were divided by the total signals and reported as a percentage: %Dimer crosslinked =





(all crosslinked products)

I(crosslinked products) + I(m/z 931) + I(other products)

×100 where I(other products) includes signals corresponding to water and ammonia and other loses/fragments, and I(m/z 931) is the prod-

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219

Fig. 1. (a) MALDI–mass spectrum of angiotensin II before pyrolysis, (b) MALDI–mass spectrum of the non-volatile pyrolysis products at 220 ◦ C of angiotensin II. (Inset 10× intensity). (c) MALDI–mass spectrum of the non-volatile pyrolysis products at 270 ◦ C of angiotensin II (Inset 40× intensity). See Table 1 for peak assignment.

uct due to cleavage at aspartic acid. From the plot in Fig. 2 it can be determined that approximately 25% of the signals can be attributed to the cleavage at D. Similarly, approximately 20% of the signals can be attributed to the formation of the dimmer crosslinked product.

For both the cleavage at D and the formation of crosslinked peptides, a maximum product yield for both processes is observed at temperatures between 240 and 260 ◦ C. Also from this plot one can observe that there is no definite temperature where one process

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(e.g., cleavage at D) is specifically enhanced over the other (e.g., peptide crosslinking), indicating that the energetics of the processes are similar. To determine the sequence of the crosslinked peptide, a tandem mass spectrum of the [M+H] + ion at m/z 1942 was performed and it is shown in Fig. 3. Inspection of the tandem mass spectrum indicates that the ion at m/z 1942 is likely formed by the fusion of the two peptides. That is, the crosslink product is a mixture of RVYIHPF–DrVYIHPF and DrVYIHPF–RVYIHPF, as both fragment ions corresponding to each of these combinations were detected. The large signals observed for the fragment ions y7 and b8 can be attributed to a mixed population of singly protonated ions of sequence DrVYIHPF–RVYIHP. That is, one ion is protonated at the Nterminus of the peptide and would lead to the strong b8 ion signal, while the strong y7 ion signal derives from the peptide singly protonated at the arginine side chain. No other fragment ion is observed in this tandem mass spectrum most likely due to the internal arginine group sequestering the proton, thus preventing its mobility along the peptide backbone and in turn an extensive fragmentation of the peptide [13]. Multistage tandem MS analysis (MS3 , shown in Fig. 4) was also carried out on the ion at m/z 931, and signals match the expected sequence for the peptide resulting from the cleavage at D of angiotensin II.

30 Condensation products / Total products D-cleavage products / Total products

25

Percentage (%)

20

15

10

5

0 180

200

220

240

260 270 280 290 300

Temperature (ºC) Fig. 2. Temperature dependence of peptide condensation and D-cleavage processes for the peptide angiotensin II (Sequence: DRVYIHPF). See text for calculation of the percentages (error bars represent ± 1 standard deviation, n = 5).

b1 116

b2 255

b3 354

b4 517

D r V Y b1 157

y14 1827

y13 1688

y12 1589

b2 256

b3 419

b4 532

R V Y y14 y13 1786 1686

35000

b5 630

b6 767

I

H P F

y10 y11 1426 1313 b5 669

I

b8 1011

b7 864

b6 766

y9 1176

b9 1168

y8 1079

y7 y6 931 775

b9 1168

H P F D

r

y11 y12 y10 1524 1411 1274

y9 1177

b11 1430

y8 1029

y7 914

b10 1267

H P F

y5 676

y4 513

b11 1430

b12 1543

V Y I y6 775

y5 676

b14 1777

b13 1680

b12 1543

R V Y I

b8 1028

b7 913

b10 1267

y3 400

y2 263

y1 116

b14 b13 1680 1777

H P F

y4 513

y2 y1 263 116

y3 400

5000

y 30000

4000

25000

3000

7

b

8

1941

931 1011

Intensity

1942

20000

2000

1924

b 15000

1000

b

8

1028

y

1029

10000

13

1680

8

y

14

1786

0

600

800

5000

1000

1200

1400

1600

1800

1200

1400

1600

1800

1901

931 1011

0 600

800

1000

2000

m/z Fig. 3. ESI-tandem mass spectrum of the ion at m/z 1942 (Inset 5× magnification). Ions with red labels are matched to a peptide of sequence DrVYIHPFRVYIHPF (where r = Arg–NH3 ). Ions labeled in black are matched to a peptide of sequence RVYIHPFDrVYIHPF. Ions labeled in blue are matched to both peptide sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 5. ESI-mass spectrum of the pyrolysis products of an equimolar mixture of the peptide Leu-enkephaline (YGGFL) and the peptide HA (YPYDVPDYA). The condensed product peak is observed at m/z 1640.

Fig. 4. Multistage tandem MS analysis (MS3 ) of the condensed product of angiotensin II at m/z 1942 (1942 → 931 → products).

To further investigate the formation of pyrolysis peptide crosslinked products as well as their structures, equimolar amounts of the peptides Leu-enkephalin (sequence: YGGFL; M1 ) and the HA peptide (sequence: YPYDVPDYA; M2 ) were pyrolyzed at 220 ◦ C. The b2 221

b1 164

Y

b4 452

b3 278

G G F y13 1477

y12 1420

y11 1327

b3 424

b4 539

Y P Y

D

b1 164

b2 261

y13 1477

y12 1380

y11 1217

b5 538

L

non-volatile products were analyzed by direct infusion ESI–MS. The resulting mass spectrum is shown in Fig. 5, where a signal at m/z 1640.3 is observed and corresponds to the protonated molecule of the condensation of the two peptides. Further analysis by MS/MS

b7 798

b6 701

b8 962

Y P

y10 1216 b5 638

y9 1102 b6 735

V y10 1102

Y y8 939

b7 850

P

b9 1077

b10 1176

b11 1273

D

V

P D

y6 679

y7 842 b8 1013

D

y9 1002

221

A

y7 790

y8 905

Y y5 556

y6 627

y9

35000

b10 1248

b9 1084

Y

y5 556

1102

Y

y4 465

y3 368

b11 1305

b12 1362

G G y4 393

b13 1551

b12 1388

y3 336

A y1 90

y2 253 b13 1509

F

L

y2 279

y1 132

b10 1176

30000

25000

Intensity

b9

b12

1077

1388

20000

b10-H2O

1013

939 y9 1002

b4

1551

850

638

b9 b8

539

b13

1158

b7

b5

10000

5000

b8

y8

15000

1084

962

y11

y12 1380

1639 1622 1640

1217

0 600

800

1000

m/z

1200

1400

1600

Fig. 6. ESI-tandem mass spectrum of the ion at m/z 1640. Ions with red labels are matched to the peptide sequence YGGFL-YPYDVPDYA. Ions labeled in black are matched to the peptide sequence YPYDVPDYA-YGGFL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Scheme 1. Proposed mechanism for intermolecular peptide condensation involving nucleophilic attack to the C-terminus carbonyl group of peptide M1 by the N-terminus amine group of peptide M2 .

of the ion at m/z 1640 was conducted and results are shown in Fig. 6. Inspection of the tandem mass spectrum indicates that the ion at m/z 1640 was most likely formed by the permutated condensation of the two peptides, that is [M1 + M2 –H2 O + H]+ and [M2 + M1 –H2 O + H]+ . Fragment ion data show that the crosslink product is a mixture consisting of the peptides of sequence YGGFL–YPYDVPDYA and YPYDVPDYA–YGGFL, as both fragment ions corresponding to these peptides were identified in the tandem mass spectrum in Fig. 6. Detailed analysis shows characteristic

intense ions due to fragmentations of peptide bonds near proline (e.g., b10 and y8 ) and aspartic acid (b9 and b12 ) in Fig. 6. Combined, these data conclusively shows that peptide crosslink products are formed during the pyrolysis of peptides. The data presented so far points to a preferential C-terminus to N-terminus condensation between peptides. This trend may be driven by a combined effect of steric hindrance and the pKa ’s of the nucleophile groups. The difference in pKa values between the N-terminus amine (pKa = 9.6) and the side chain amine group in arginine (pKa = 13.2) can explain the preferential C to N-terminus bonding as the amine group in arginine is most likely to be protonated if the peptide was derived from an acidic solution, making this side chain protonated amine group a poor nucleophile. Also, if a neutral peptide is considered the high pKa of the side chain amine would hinder proton transfer necessary for complete nucleophilic attack to the carbonyl carbon. Scheme 1 shows the proposed mechanism of nucleophilic attack to the carbonyl group by the amine group. In step b of this mechanism a proton transfer is required from the amine nitrogen to the carbonyl oxygen. However, if the amine group has a higher pKa as in arginine, it will sequester the proton and hinder its transfer. To further investigate the chemical moiety involved in the intermolecular fusion between peptides, an experiment was conducted where a N-terminus acetylated angiontensin II was pyrolyzed at 220 ◦ C. Analysis of the non-volatile products by MALDI–MS showed no detectable signals corresponding to condensation and/or crosslink products, with only the loss of water peak detected (Fig. 7a). Pyrolysis of this N-acetylated peptide at a higher temperature of 270 ◦ C also failed to produce any crosslink product, as illustrated in the MALDI–mass spectrum of the same sample (Fig. 7b). Interestingly, the N-acetylated angiotensin II peptide did not undergo cleavage at aspartic acid (indicated by the product peptide at m/z 931), the causes which are currently being investigated in our laboratory.

4. Conclusion

Fig. 7. MALDI–mass spectra of the pyrolysis products of N-Ac-Angiotensin II at (a) 220 ◦ C, (inset 200× magnification), and at (b) 270 ◦ C (inset 40× magnification). Both analyses show no detectable condensation products when the N-terminus of the peptide is acetylated.

The hetero and homo intermolecular condensation resulting from the pyrolysis of peptides were observed by MS analysis of the non-volatile pyrolysis products. Tandem MS analyses demonstrated that intermolecular C-terminal to N-terminal bonding is favored during pyrolysis when forming a bond between two peptides, rather than involving basic and acidic side chain groups like arginine and aspartic acid. Further evidence of N-terminus to Cterminus condensation was provided by the pyrolysis of a standard N-acetylated peptide containing an internal arginine group, which did not form any detectable crosslinked product. Peptide crosslink-

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ing during pyrolysis of proteins and peptides has been shown to occur at pyrolysis temperatures between 180 and 300 ◦ C. Even though sequence information of the individual peptides involved in the condensation reaction is preserved, caution must be taken in analyzing sequence data derived from MS/MS analysis of condensed peptides as these result from permutated combinations of the reacting peptides, resulting in the scrambling of sections of the original peptide sequence [14]. The implementation of a multistage tandem MS analysis (e.g., MS3 ) could circumvent this shortcoming by analyzing shorter fragments of the condensed peptide that would accurately correspond to a sequence of the original peptide and/or protein. Studies are currently underway to explore these condensation reactions in the thermal decomposition of high molar mass proteins. Acknowledgement The authors are thankful to the National Science Foundation (NSF-CAREER Award CHE-0844694) for support and funding of this project. References [1] J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Electrospray ionization for mass spectrometry of large biomolecules, Science 246 (1989) 64–71. [2] N.B. Cech, C.G. Enke, Practical implications of some recent studies in electrospray ionization fundamentals, Mass Spectrom. Rev. 20 (2001) 362–387.

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