A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

Abstract

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

“…Resulting MALDI-MS spectra thus contain both kinds of peaks, the peptide chain’s MH + reduced by ISD, as well as the disulfide bonded peptide (DSB-peptide) precursor ion, which are all subsequently fragmented by MS/MS in the TOF/TOF ion optics. 53 The reduced peptides are directly identified by in-sequence search of the target protein, and the disulfide bond connectivity is assigned based on the triplet rule with the prerequisite that both reduced and DSB-bonded peptides coelute in the LC. False-positives are minimized in the qualified candidates list, as peptide sequences that do not contain cysteine residues are automatically filtered out.…”

Nanotechnologies in proteomics

“…Upon application of this scheme, the additional knot LIFTraises the potential energy of the ions after TOF1 analyser; second accelerator stage is realized in TOF2. This approach provides high performance of automated MS and MS/MS analyses and high sensitivity -as was demonstrated for protein from lysate of human endothelial cell line [83].…”

The reproducibility of phospholipid analyses by MALDI-MSMS

“…8,17 When fragmentation occurred by CID in a triple quadrupole a range 80 of outcomes resulted. These included: no evidence for cleavage of the acyl chains; 11 evidence of the loss of the acyl chain from each position with a statistical difference in their intensities; 10,11,13,22 evidence of the loss of the acyl chain from each position with no statistical difference in their 85 intensities; 19 and evidence of the loss of each chain as different types of fragments, with differences in the relative of a collision gas, 26 and using Collision Induced Dissociation (CID) in the presence of argon. The benefits of the addition of a lithium salt during the MALDI TOF/TOF analysis of lipids are reported, along with the relative intensities of the product ions corresponding to the loss of the acyl chains from the sn-1 25 and sn-2 positions of the lithiated and protonated molecular ions, determined from a number of automated analyses.…”