Investigation of VUV Photodissociation Propensities Using Peptide Libraries

Abstract

PSD does not usually generate a complete series of y-type ions, particularly at high mass, and this is a limitation for de novo sequencing algorithms. It is demonstrated that b(2) and b(3) ions can be used to help assign high mass x(N-2) and x(N-3) fragments that are found in vacuum ultraviolet (VUV) photofragmentation experiments. In addition, v(N)-type ion fragments with side chain loss from the N-terminal residue often enable confirmation of N-terminal amino acids. Libraries containing several thousand peptides were examined using photodissociation in a MALDI-TOF/TOF instrument. 1345 photodissociation spectra with a high S/N ratio were interpreted.

Figure 1

Photodissociation (PD) and postsource decay (PSD) spectra of EGADLSIVTR.

Figure 2

b₂, b₃ ions and complementary x- and y-type ions in the photodissociation (PD) and postsource decay (PSD) spectrum of NLADLGIQLR.

Figure 3

Normalized intensities of (a) b₂ ions; (b) b₃ ions with various sequences and different residues to provide amine groups for cleavage bonds. In square brackets, the numbers specify the number of [identified b₂ ions/identified peptides/peptides in libraries] respectively.

Figure 4

The number of spectra containing various of false b/x ion pairs. (a) b₂/x_N-2 ion pairs; (b) b₃/x_N-3 ion pairs

Figure 5

Comparison of the accuracy of identifying b₂ and b₃ fragments before and after recalibration of spectra using y₁ ions.

Figure 6

Assignments of (a) v_n+1 radical ion for EGAILSIVLR; (b) v_n, w_n ions of DAIQQAFGAR; (c) v_n+1 radical ion of QLVGQVAANVR; (d) v_n, w_n ions of NGAYLGIQTR; (e) v_n, w_n and v_n+1radical ions of TVQALGLR; (f) v_n, w_n) and v_n+1radical ions of SLDLDSIIAEVK^g. * shows the isotopic peaks and ^g means that K is guanidinated.

Figure 7

Immonium ions and internal fragments from the low-mass region of photodissociation spectra of (a) ELAYHEIQLR; (b) YQEVGPNPSER. Highlighted residues correspond to immonium ions and underlined sequences label internal fragments.

Figure 8

High-mass regions of photodissociation spectra of (a) ETAILSIVLR; (b) ELAILSPVLR; (c) NLADHEIVLR.

Figure 9

Y-type ions in photodissociation spectra of (a) FQAAAGQLAQPHR; (b) YQEHLSNPFER; (c) EGAYLGPVLK^{g. g} means that K is guanidinated.

Scheme 1

Pathways to form various fragment ions: (a) v- ions; (b) w ions; (c) v_N+0.1 and v_N ions.

Scheme 2

Pathways to form w_N and v_N from (a)–(b) aspartic acid and (c)–(d) asparagine

Scheme 3

Proposed pathways to form v_N+2 ions from peptides with N-terminal (a) threonine; (b) serine.