Large-scale analysis of peptide sequence variants : the case for high-field asymmetric waveform ion mobility spectrometry

[Image: see text] Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX(K/R) and SXPFKXpSPLXFG(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC–MS/MS alone identified 8% and LC–MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library

Assessing Intrinsic Side Chain Interactions between i and i + 4 Residues in Solvent-Free Peptides: A Combinatorial Gas-Phase Approach †

Ion mobility measurements and molecular modeling techniques have been used to survey the gas-phase structures of a series of alanine-rich peptides. The peptides, examined as [M + 2H] 2+ ions, have the general forms NH 2 -(Ala) 7 -Xxx-(Ala) 3 -Yyy-(Ala) 3 and Ac-(Ala) 7 -Xxx-(Ala) 3 -Yyy-(Ala) 3 , where residues 8 and 12 are randomized. In total, 160 different peptide ions (80 related NH 2 -terminated and -acetylated sequences) have been studied. Substitutions of residues 8 and 12 permit an assessment of the influence of specific interactions between residues in an adjacent helical turn. The formation of helices and globular structures in the gas phase appears to be sensitive to specific interactions between amino acid side chains. A preliminary discussion of these results in terms of what is currently known about helix formation in the gas phase and in solution is given. Overall, it appears that this combinatorial approach to studying sequence-tostructure interactions that are intrinsic to the peptide is a viable strategy for surveying trends in large numbers of sequences without interference from solvation effects

Formation of peptide aggregates during ESI: size, charge, composition, and contributions to noise

AbstractIon mobility/time-of-flight techniques have been used to examine the onset of aggregation in model systems of Gly-Xxx (where Xxxx = Ala, Asn, Asp, Gln, Glu, His, Leu, Ser, Thr, and Trp) dipeptides. Under the experimental conditions employed, there is evidence that simple binary and quaternary mixtures of these dipeptides produce clusters containing as many as 16 to 75 peptide units (and 1 to 7 charges). In some systems, cluster compositions appear to come about largely from statistical association of peptide units; other dipeptide mixtures (and generally for small clusters) show evidence for nonstatistical behavior which could arise from some differences in gas-phase or solution thermochemistry. The minimum aggregate size appears to be largely determined by the charge state. Average thresholds for aggregate formation in the z = 2, 3, and 4 charge state families occur at m/z ∼500, 660, and 875, respectively. We briefly consider the idea that aggregates formed during electrospray ionization (ESI) may contribute to the background signal observed in the analysis of complex peptide mixtures