Mass spectrometric analysis of modified proteins and peptides

Until now, following modifications of histone, p53 and PARP-1 depicted in Figure 29 have been published [143, 186, 218, 219].

H2A

H2B

H3

H4

E3 E16 E115 K213

E3

H1

K13

K30

K27 K37

K16

PARP-1 p53

D387 E488E491 E258 D259 E271

Figure 29 ADP-ribosylated residues in histones, p53 and PARP-1. Schematic representation of human histone poly(ADP- ribosyl)ation sites. The positions of covalent modification sites (branched structures) of human histones H1 and H2B have been predicted from corresponding rat coordinates [143, 186, 218, 219]. Schematic representation of human p53 poly(ADP-ribosyl)ation sites and PARP-1 [50]. Figure adapted from [220, 221].

To confirm that modified NAD⁺ compound: 2,3”mNAD⁺ can be used to find the places of modification many problems need to be solved. One of this is to apply suitable mass measurement methods to be able to detect modifications, moreover to optimize the reaction to obtain heigh yield of modified protein and purification methods to separate modified from unmodified protein.

69 The molecular weight (MW) of histone H1.2, [M+H]⁺calc 22056,4, was confirmed by ESI-MS and MALDI-TOF-MS, giving the following results:

[M+H]⁺exper 22054,8; 22058,3. In the case of protein histone H2B, [M+H]⁺calc(Av)14597.80, the following results were obtained: [M+H]⁺exper14595.2;

14598,8. Thus, the calculated and experimental molecular weights were in good agreement. Unfortunately, it was not possible to determine by mass spectrometry the MW of the modified proteins, neither histone H1.2 nor histone H2B. The main problem was the purification of the reaction mixture, the presence of buffer and hPARP1 protein that disturbed the MS analysis. When the samples were diluted and analyzed by ESI-MS, yet no m/z signals of proteins but merely those of detergent-like substances were detected. Even after desalting the samples using C4 ZipTips, the proteins could not be detected by mass spectrometry. In order to remove the interfering compounds, the entire sample content was precipitated by TCA, washed with cold acetone, centrifuged, dried and then reconstituted in 5 μl hexafluoro-2-propanol (HFIP). A volume of 1 μl of the resulting solution was subsequently diluted with 20 μl of 0.1 % formic acid (FA)/50 % MeCN and analyzed by ESI-MS. After this sample preparation procedure, the proteins could be detected, yet they were not modified. A disadvantage of the protein precipitation method is that it is not certain that the modified protein will precipitate. It is expected that the sample consists of modified and unmodified protein. A possible approach would be to purify the protein mixture by HPLC on a C4 column; however, large amounts of proteins are needed. Moreover, the open question is how much of modified protein is obtained in the PARylation reaction, when using modified NAD⁺ as a substrate. Histone H1.2 wt was digested with AspN (Figure 30) in solution, (gel A line 2) and in gel, (gel B, line 1).

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Figure 30 Digested Histone 1.2 with AspN. (A) (M) marker, (1) H1.2 wt, (2) digested Histone 1.2 in solution for 3 h at 36 °C; (B) (M) marker, (M) low molecular weight marker; (1) Tris tricin gel stained with Coomassie Blue H1.2 w.t digested in gel; (2) H1.2 mod digested in gel (3) H1.2 wt; (C) The same gel as in (B) in fluorescent readout.

These results indicated that the digestion of histone H1.2 in solution proceeded more efficient than in gel. Unfortunately, in the case of in-solution digestion, the presence of buffer and hPARP-1 had a negative influence on the mass spectrometric analysis of histone H1.2 proteolytic fragments. Dialysis could be used to purify the histone H1.2. Very efficient would be to purify the reaction mixture by HPLC; however, this separation method requires a large amount of protein. Nevertheless, the modified peptides could be seen on tris-tricine gel in the fluorescent readout (Figure 30C).

Digested histone H1.2 was subjected to mass spectrometric analysis, giving 52 % sequence coverage (Figure 31). Unfortunately, it was not possible to identify the modification when modified histone H1.2 was digested and further analyzed by MS.

Figure 31 Histone H1.2 which was subjected to in-gel digestion with AspN and mass spectrometric analysis; sequence coverage 52 %.

Moreover, histone H1.2 as well as modified histone H1.2 was digested with trypsin. Database search was performed using the ProteinLynx Global

71 Server (SwissProt, all species) and Mascot (UniRef100, all species) search programs. (PLGS results are essentially the same). Unfortunately, no modified peptide could be identified. The remaining solution was desalted and concentrated by ZipTip C18. The sample was mixed 1:1 with matrix solution (0.7 mg/mL HCCA in 85 % ACN, 0.1 % TFA) and spotted on the MALDI-target. The MALDI/MS/MS measurement was also performed automatically. The provided amino acid sequence was compared to the digested peptide teorethical pattern.

The w.t. sample was compared to the modified samples. The following proteolytic peptides found only in the modified samples were subjected to MS/MS fragmentation. No peptide containing the putative modification in either case could be identified. Furthermore, histone H1.2 as well as histone H2B was modified and digested in gel with Glu-C and transferred to autosampler vials for LC/MS/MS. A volume of 5 μl was injected. Database search was performed using the Mascot (SwissProt, all species) search program. Unfortunately, no modified peptide could be identified.

The best from the sequence point of view protein known to be modified by PARP-1 is p53 since it contains not so much lysine aminoacids as histones.

This protein was digested with trypsin, pepsin, AspN, followed by mass spectrometric analysis of proteolytic mixtures. Sequence coverage of 58 % was obtained (Figure 32).

Figure 32 p53 digested with trypsin, pepsin, AspN, 58.27 % coverage.

Modified protein p53 was digested in gel with trypsin at 60 °C, 34 min in microwave. The supernatant was removed and then the proteolytic peptides were extracted from the gel pieces with 150 µL 0.1 % TFA/50 % acetonitrile for 15 min at 40 °C in an ultrasound bath. The two supernatants were combined and dried. The sample was dissolved in 15 µL 0.1 % TFA. A volume of 1µL

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desalted sample was placed on anchor chip target. The sample was covered with matrix solution (0.7 mg/mL α-cyano-4-hydroxycinnamic acid (HCCA) in 85 % ACN, 0.1 % TFA, 1mM NH4H2PO4), 1:1 v/v. The provided amino acid sequence (GST-p53) was compared to the digested peptide pattern. After Trypsin digestion, all 3 samples showed the following peptide. Range Mono MH⁺ Partials Sequence [268-273] 751.373 NSFEVR. The amino acid sequence was confirmed by MS/MS. The sample GSTp53wt showed also the following peptide. Range Mono MH+ Partials Sequence [249-267] 2068.176 RPILTIITLEDSSGNLLGR. The amino acid sequence was confirmed by MS/MS.

There were over 30 signals present only in the modified samples. But the MS/MS spectra of these signals could not be assigned to the amino acid sequences.

For 2,3”mNAD⁺ the ions with m/z 740 were found and fragmented by MS/MS. About 30 signals (MS/MS spectra) which were present only in the modified samples were compared to the fragmention pattern of 2,3”mNAD⁺. But no analogy has been found.