Peptide De Novo Sequencing With MALDI TOF/TOF: A Simple Approach Using Sulfonation Chemistry

One of the main focuses of the scientific community, now that major advances have been achieved in the comprehension of genome organization, has become the study of proteins, including their structure and activities. Among the different techniques available, mass spectrometry has played a fundamental role and is experiencing rapid growth.1Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF)/TOF instruments (Ultraflex Bruker Daltonics, Bremen, Germany), taking advantage of the reflectron mode, combine a soft ionization process with good sensitivity and resolution.2

Until recently, many proteomic projects were aimed at identifying proteins in biological samples in order to characterize the proteomic profile of subcellular compartments, cells, tissues, or pathological conditions. A deeper comprehension of the protein machinery will shed light on the complex and highly dynamic protein network (interactome), how this network transduces external stimuli inside the cell (signalosome), and how it regulates cellular processes (metabolome). Moreover, the identification of biomarkers for severe pathological conditions, such as cancer, heart disease, and neurodegenerative diseases, will allow the establishment of early diagnosis and the detection of pharmaceutical targets.

Experimentally, in peptide mass fingerprinting (PMF),3 proteins, after enzymatic digestion, are resolved in a number of peptides, whose masses are determined and matched with a sequence database. However, the presence of splicing variants in cells, combined with the generation of different protein isoforms or fusion proteins, gives rise to a complex picture that demands a more detailed analysis. Furthermore, the study of species with yet uncharacterized genomes or the investigation of post-translational modifications (PTMs) is not possible with classical PMF, requiring simple, reliable de novo sequencing. In the last 5–7 years, much effort has been expended in order to improve the performance of peptide sequencing with MALDI-TOF instruments.4

Described here is a fast, robust chemical modification of peptides that strongly improves de novo sequencing, using CAF™ (Chemical Assisted Fragmentation) chemistry (GE Healthcare/Amersham Bioscience, Uppsala, Sweden).

Principles and technical procedures

In MALDI instruments, postsource decay (PSD)5 produces nearly random fragmentation of the peptide backbone, generating mainly b- and y-ions, but other ions as well. In order to improve the peak intensity and obtain easily interpretable spectra (i.e., only one series of ions), a chemical compound carrying a sulfonation group is used.6–8 This group reacts specifically with primary amines of peptide chains (Figure 1b). In the first step (Figure 1a), ε-amino groups of lysines are blocked, leaving the N-terminal amino group the only one able to be sulfonated. After reaction with the CAF reagent, a sulfo group is transferred to the N-terminus. During the ionization process, two protons are captured by each peptide (Figure 1c). Postsource peptide fragmentation generates b- and y-series fragments; the y-series, carrying one proton, possesses a single positive charge and will reach the detector; however, the b-series, which carries the negative charge of the sulfo group at the N-terminus, after taking up the second proton, becomes neutral and is therefore not detected (Figure 1c). In this way, a single and often complete series of y-ions is present in the final spectra, leading to easy, safe interpretation of the sequence.

Figure 1 a) - Work flow of de novo sequencing with CAF chemistry. b) Molecular formula of N-terminal sulfonation. c) Schematic representation of charge distribution after peptide sulfonation. The sulfo group generates a negative charge at the N-terminus. During the ionization and fragmentation processes, two series of fragments, mainly b- and y-type, are produced, and both capture a proton from the matrix. In the N-terminal fragment (bseries), the positive charge of the proton is counterbalanced by the negative charge of the sulfo group, generating neutral molecules that are unable to reach the detector; only the C-terminal fragments (y-ions) carry a positive charge and will travel in the mass spectrometer.

Proteins to be analyzed were separated by polyacrylamide gel electrophoresis and stained with appropriate methods. Selected spots were cut out, destained as previously described,9 and incubated with trypsin. This generates peptides with basic amino acids (lysine or arginine) at the C-terminus, as required for efficient fragmentation in the following steps. The sulfonating reagent reacts with all primary amino groups; in order to analyze lysine-ending peptides, it is therefore necessary to block the ε-amino group of lysines, leading to peptides labeled with only one sulfonic group (i.e., at the N-terminus). At least two methods are available: 1) the addition of O-methylisourea hydrogen sulfate (17.2 mg/mL in 0.25 M NaHCO3, pH 10) and incubation overnight at room temperature (RT), which converts lysine into homoarginine in a guanidination reaction (adding 42 Da/Lys), thus eliminating the reactive ε-amino group, and 2) incubation of the peptide mixture with 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag 4H, Agilent Technologies, Palo Alto, CA) for 3 hr at 55 °C (adding 68 Da/Lys).

Peptide derivatizations were performed on solid-phase supports: A μZipTip™ C18 (Millipore Corp., Bedford, MA) was wetted with a solution of trifluoroacetic acid (TFA)/60% acetonitrile, then equilibrated with 0.1% TFA. The peptide solution, deprived of any organic solvent, was pipetted up and down approx. 10 times in order to adsorb the peptides onto the reversed-phase material. After column washing with 0.1% TFA, the freshly prepared labeling solution (1 mg/10 μL in 0.25 M NaHCO3, pH 9.4) was slowly pipetted up and down, and allowed to react with the adsorbed sample for 3 min at RT. The reagent was then washed out with 0.1% TFA, and a solution of 5% hydroxylamine in the same labeling buffer was pipetted up and down a few times in order to remove unspecific binding of the CAF reagent onto hydroxyl-containing amino acid residues. The sulfonated peptides were eluted by drawing 2–5 μL of 0.1% TFA/60% acetonitrile up and down the tip. The sulfonation adds 136 Da to primary amines in the peptide. A peptide with C-terminal Arg will increase its mass by 136 Da, while peptides with a C-terminal Lys will increase by 136 + 42 = 178 Da for guanidation Lys blocking, or 136 + 68 = 204 Da for imidazole incorporation (additional internal Lys residues take up 42 or 68 Da each). If a Lys-containing peptide was sulfonated without the blocking step, it would be sulfonated also on the ε-amino group.

Figure 2 a) - PMF of a tryptic digest of cytochrome C from Candida krusei. b) The same peptide mixture shown in (a) was Lys-blocked with imidazole (addition of 68 Da) and labeled at the N-terminus with the sulfo group (addition of 136 Da). The MALDI spectrum after these modifications shows more peaks and higher intensity compared to the native spectrum in (a). c) De novo sequencing of the peak 983.464 (779.425 + 68 + 136). PSD of this peptide produces a neat series of only y-ions. The bordered numbers show the difference in daltons between two adjacent peaks: These masses fit with good precision with the theoretical masses of the corresponding amino acids.

For MALDI TOF/TOF analysis, samples were prepared with the dried-droplet method: 0.3 μL of the sample was mixed with an equal volume of a saturated solution of ε-cyano-4-hydroxycinnamic acid (HCCA, Bruker Daltonics, Bremen, Germany) in 0.1% TFA/40% acetonitrile; a 0.3-μL drop was deposited on the polished stainless steel MALDI target. All samples were analyzed in reflector mode before and after derivatization to obtain PMF spectra. By comparing these spectra and scanning for additions of 136 or 178 (204) Da, candidates for sequence analysis were identified. The instrument was then switched to PSD mode and the ion selector was set to the m/z values of the precursor ions with a window ±0.2–1% of the parent ion mass.

Results

In order to test the efficiency of the CAF chemistry, the authors used cytochrome C from Candida krusei. After trypsin digestion and PMF, a number of peptides are present in the spectrum (Figure 2a). The peptide mixture was then blocked at lysines with the imidazole compound (addition of 68 Da per lysine), followed by CAF modification. The presence of the imidazole group renders the peptides more basic, thus enhancing both the number of peptides detected and the intensity of the peaks (Figure 2b). As an example, the authors selected the peptide of 983.46 Da (see arrow in Figure 2b), arising from the diminutive peak of 779.42 Da in Figure 2a (see arrow). PSD fragmentation of the peptide gives rise to a clear series of only y-ions, generating a clear and definitive amino acid sequence (Figure 2c).

Figure 3 a) - Peptide mixture of an unknown protein from the mollusk Mytilus galloprovincialis was Lys-blocked by guanidation, followed by sulfonation of the N-terminus. The figure shows the MALDI spectrum of the modified peptides. b) PSD of the peptide of 2201.968 Da (see arrow in [a]). The complete sequence of 17 amino acids is easily readable.

Although recent advances in the study of genomes have made complete DNA sequencing of several species available, the majority of them are still uncharacterized. For all of these species, in the absence of a complete and reliable genome database, identification of proteins with normal PMF is impossible. Positive and unambiguous de novo sequencing is a useful approach for proteomic studies of this type of sample. In one proteomic study, the identification of proteins from the bivalve mollusk Mytilus galloprovincialis was required. After in-gel trypsin digestion, matching the peptide list with databases did not provide any protein identification due to lack of data for this species (data not shown). The authors overcame this problem by selecting representative peptides for de novo sequencing. Peptides were first lysine-blocked with the guanidination reaction, and then sulfonated on the N-termini. Figure 3a shows the MALDI spectrum of one of these sulfonated proteins. Figure 3b represents the case of the peptide of 2002 Da (see arrow in [a]); it should be noted that this peptide has a peak of quite low intensity in the MALDI spectrum in comparison to other major peaks. PSD fragmentation generated a clear sequence of 17 amino acids (SL/IEPEEMQEVI/LDAMFEK); a search for homology with this sequence in the available database permitted the identification of the protein as a cAMP-dependent protein kinase. Hence, this approach allows the identification of unknown proteins by sequence homology, which is error-tolerant compared to PMF.

Conclusion

Increased proteomic interest has expanded the demand for protein sequence information. To this purpose, improved PSD after N-terminal sulfonation provides a powerful and easy tool, suitable for varied experimental purposes such as confirmation of PMF protein identification, study of the proteome from uncharacterized species, and analysis of PTMs.

References

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The authors are with the Ludwig Institute for Cancer Research, Box 595, SE-751 24 Uppsala, Sweden; tel.: +46 18 160423; fax: +46 18 160420; e-mail: [email protected]. This work was partly supported by fellowships to P. Conrotto from Associazione Italiana per la Ricerca sul Cancro (AIRC). The authors thank Dr. Antonio Villamarin, University of Santiago de Compostela Lugo, Spain, for providing samples from the mollusk Mytilus galloprovincialis.