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Review
. 2015 Jan-Feb;34(1):43-63.
doi: 10.1002/mas.21406.

Lessons in de novo peptide sequencing by tandem mass spectrometry

Katalin F Medzihradszky, Robert J Chalkley
Review

Lessons in de novo peptide sequencing by tandem mass spectrometry

Katalin F Medzihradszky et al. Mass Spectrom Rev. 2015 Jan-Feb.

Abstract

Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are "translated" into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general.

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Figures

Figure 1
Figure 1
Peptide fragment ions [Biemann, 1990]. The occurrence and mass calculations of these fragments are presented in Tables 2 and 3.
Figure 2
Figure 2
Sequence of the protocatechuate 3,4-dioxygenase type II beta subunit PcaH-II [Agrobacterium tumefaciens]. Two tryptic peptides (underlined) were identified in database searches of both of the digests.
Figure 3
Figure 3
Low-energy CID spectrum of m/z 557.8(2+). The corresponding sequence was determined from this spectrum as Ser-Gly-Asn-Phe-Ser-Phe-Gln-Thr-Val-Lys.
Figure 4
Figure 4
Sequence of protocatechuate 3,4-dioxygenase type II alpha subunit PcaG-II [Agrobacterium tumefaciens], NCBI# 11037227. The underlined sequence is homologous to the tryptic peptide sequenced from the spectrum in Figure 3, with S->N, T->S and H->Q substitutions in positions 3, 5, and 7, respectively.
Figure 5
Figure 5
Low-energy CID spectra of the tryptic peptide SGNFSFQTVKPGR, from the triply- charged precursor (upper panel) and the doubly-charged precursor (lower panel).
Figure 6
Figure 6
Low-energy CID spectra of m/z 684.7(3+)(top), 690.0(3+) (middle) and 703.7(3+) (bottom). These spectra represent tryptic peptide, VPTADGVMQAPHLALSIFGK unmodified, with a Met-sulfoxide, and with a carbamidomethyl Met, respectively. The spectra are presented in reverse elution order.
Figure 7
Figure 7
Low-energy CID spectrum of m/z 656.8(2+), representing the C-terminal tryptic peptide of the protein.
Figure 8
Figure 8
The manually deciphered sequence of sulfocatechol 3,4-dioxygenase alpha-subunit of Novosphingobium resinovorum (Sphingomonas subarctica) (NCBI # 56787886) is shown in the upper panel. The genomic sequence was determined later utilizing this information, as presented in the lower panel. The correctly determined sequence of a tryptic peptide that did not show sufficient similarity to the “template” and thus its sequence position could not be predicted is printed in bold.
Figure 9
Figure 9
Low-energy CID spectrum of a chymotryptic-type peptide; precursor at m/z 729.9(2+). A fragment assignment comparison for the different sequence solutions proposed by the PEAKS software (Supplement 3) from manual sequencing (Table 5), and for the correct sequence (Figure 8) is presented in Supplement 5.
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