“De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study

Abstract

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066 -5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-tonoise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process. T he use of mass spectrometry combined with database searching has gained wide acceptance for rapid, sensitive protein identification [1]. This method requires the availability of extensive protein, cDNA, or genomic sequence from the organism of interest (or at least from a closely related species). If such sequence information is not available, as remains the case for the vast majority of extant organisms, it is desirable to utilize de novo peptide sequencing to identify and obtain information about their protein(s). Although Edman sequencing has for a long time been a method of choice for de novo sequencing of peptides and proteins, mass spectrometry (MS) has also been shown to have considerable utility for this purpose.Indeed, mass spectrometric de novo sequencing based on the fragmentation of derivitized peptide ions dates back to the late 1960s and on underivatized peptide ions to the early 1980s (reviewed in [2] and [3]). More recently, MS sequencing has been given considerable impetus by the development of ESI and MALDI, ionization techniques whose efficiencies for producing intact peptide ions are far higher than those that were previously available. Again, both derivitized and underivatized peptides have been used in this newer work. Thus, chemical derivatization has been applied to simplify/enhance the fragmentation spectra and/or to differentiate N-from C-terminal fragment ions [4 -13]. Notable in this regard is the use of 18 O i...

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

“…MS/MS spectra generated by MALDI quadrupole TOF mass spectrometry of singly charged peptides also showed preferential fragmentation at the N-terminal bond of Pro and at the C-terminal bond of the acidic residues Asp and Glu [8]. The same observation was made during CID analysis of singly charged peptide ions using an in-house assembled MALDI-quadrupole ion trap [9]. Several of the peptides used in our study contained Pro and/or acidic residues and some had, due to missed cleavages, internal Lys or Arg (Table 1).…”

“…The samples were allowed to air-dry at room temperature and were then inserted into the mass spectrometer and subjected to MALDI-MS analysis. Prior to analysis, the mass spectrometer was externally calibrated with a mixture of Angiotensin I, Glu-fibrino-peptide B, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH . For MS/MS experiments, the instrument was externally calibrated with fragments of Glu-fibrino-peptide.…”

“…MALDI de novo sequencing has been carried out using CID in QqTOF instruments [7,8] and by resonance excitation of singly charged peptides in a homebuilt MALDI-quadrupole-ion trap mass spectrometer [9]. In the past, limited sequence information was also obtained through the use of post-source decay (PSD) MALDI-TOF.…”

How much peptide sequence information is contained in ion trap tandem mass spectra?

“…These uninformative peaks would make identification difficult, and they are generally of lower abundance than sequence specific fragments. Therefore, spectra are frequently "filtered" so that only the most intense ions remain [18]. Depending on the application we have found a "top 4 filter" per 100 Th or a "top 6 filter" advantageous in separating signal (high intensity) from noise (low intensity).…”

“…After this the distribution of b-ions decreases until b 4 , where it stays about constant until it catches up to the number of y-ions at b 11 /y 11 . The doubly charged y-ion series starts at y 6 and continues relatively flat until y 18 . However, it is a minor number compared with either b-ions or y-ions.…”