Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry

Abstract

Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research and in studies that use photochemical crosslinking to investigate molecular aspects of protein-nucleic acid interactions. MALDI and ESI sensitivities for the two hybrid compounds were found to be similar respectively to their sensitivities for the pure oligonucleotide parts. In general, MALDI proved to be less affected by sample impurities and more sensitive than ESI, while ESI on the quadrupole produced greater mass accuracy and resolution than MALDI on the time-of-flight instrument. A hybrid's behavior in a MALDI-matrix or an ESI-spray-solvent was found to be governed mainly by the oligonucleotide. A single positive ESI tandem mass spectrum of the peptide-dT6 accounted for the heteroconjugate's entire primary structure including the point of the oligonucleotide's covalent attachment to the peptide.

Analysis of protein–nucleic acid interactions by photochemical cross‐linking and mass spectrometry

“…Nevertheless, several matrix-analyte combinations have proven to be useful for sensitive analysis of different kinds of biopolymers (for a table of commonly used matrices for different compound classes, see Jensen et al, 1996). It is apparent that MALDI matrices used for nucleic acid oligomers are not necessarily suitable for peptides and vice versa.…”

“…Because thymine has a very low affinity for fragmentation-inducing protons, poly(dT) or T-rich oligonucleotides are considerably more stable under positiveion CID MS/MS conditions than mixed base oligonucleotides (Wang, Bartlett, & Martin, 1997;Vrkic et al, 2000). The MS/MS-spectra of poly(dT)-peptide heteroconjugates published by Jensen et al (1996) and Gafken, Mosbaugh, & Barofsky (2000) demonstrate that peptide bond cleavages occur with similar yields because the fragmentation of the attached poly(dT)-oligonucleotide, i.e., sensitive peptide sequencing is possible.…”

“…Thus, MALDI conditions such as those normally applied to DNA or RNA samples, i.e., 2,4,6-THAP (Pieles et al, 1993;Bennett et al, 1994;Jensen et al, 1996;Gafken, Mosbaugh, & Barofsky, 2000) and 3-hydroxypicolinic acid (3-HPA) (Farrow et al, 1998;Steen et al, 2001) were used. In addition, it was recently demonstrated that a mixture of 2-aminobenzoic acid and nicotinic acid could be successfully applied to this type of sample (Raveh et al, 2001).…”

Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies

“…The broad applicability of soft ionization techniques, such as matrix assisted laser desorption ionization (MALDI) [10,11] and electrospray ionization (ESI) [12,13], and the ability to perform sequence determinations [14,15] make mass spectrometry (MS) the analytical platform of choice for the characterization of products obtained from probing reactions. For this reason, approaches based on crosslinking and MS detection have been successfully applied to individual substrates of proteic [16][17][18][19][20][21][22][23][24] and nucleic acid nature [25,26] and to assemblies of increased complexity involving specific protein-peptide [27,28], protein-protein [29][30][31][32][33][34][35][36][37][38][39][40][41], and protein-nucleic acid [42][43][44][45][46][47][48][49][50][51][52][53][54] interactions.…”

Collision induced dissociation-based characterization of nucleotide peptides: Fragmentation patterns of microcin C7–C51, an antimicrobial peptide produced by Escherichia coli

“…Cross-linked© peptide© or© protein-nucleic© acid© conjugates have© been© described© in© RNA© and© DNA© viruses,© in© DNA topoisomerases,©or©as©amino©acid-RNA©conjugates©in aminoacyl-tRNAs© [2].© Furthermore,© UV-induced© crosslinking© has© emerged© as© an© efficient© method© to© characterize© RNA© interactions© with© peptides/proteins,© in© particular© within© ribonucleoprotein© particles© [3,© 4].…”