1. THE APPLICATION OF A SOFTWARE TOOL FOR THE TWO-DIMENSIONAL REPRESENTATION OF LC-MS DATA TO THE ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS AND THE COMPARISON OF COMPLEX PEPTIDE MIXTURES A.Potts 1, D. Walther 2, S. Catherinet 2, W. Bienvenut 1, R. Appel 2 and M.Quadroni 1 1 Protein Analysis Facility, University of Lausanne, Epalinges, Switzerland; 2 Swiss Institute of Bioinformatics, Proteome Informatics Group, Geneva, Switzerland Contact : manfredo.quadroni@ib.unil.ch ABSTRACT A novel software tool for the visualisation of peptide LC-MS datasets as two-dimensional density maps as been developed at the Swiss Institute of Bioinformatics. The software can display faithfully both high amd low- resolution MS data, with excellent conservation of peak shapes and, thus, mass and charge state information. In addition, the tool can be used to perform comparisons of two or more datasets, in a fashion similar to classical 2D-gel analysis. We have used a prototype version of the software for two applications : 1) the detection and identification of known post-translational or artefactual modifications on (semi-)purified proteins and 2) the comparison of complex, highly similar mixtures to detect peptides and hence proteins present at low abundance which would otherwise be missed in regular shotgun LC-MS/MS analyses. We show examples of both applications using model samples as well as samples of biological origin. For the analysis of complex mixtures we have tested the approach using a digest of a complex mixture of proteins spiked with known amounts of a standard. For the analysis of PTMs we have attempted to apply the tool to study histone modifications as well as to characterize peptide modifications induced by sample treatment. We show that LC-MS data present highly reproducible 2D-patterns suitable for matching of different datasets. We also demonstrate that the software greatly facilitates the detailed analysis of these datasets and allows highlighting peptides undergoing significant quantitative variations. RT +1 ion+2 ion+3 ion Full-scan (survey scan) MS data are extracted from LC-MS or LC-MS/MS files and exported to flat text files. These are parsed by the software tool with specially adapted algorithms to sample, integrate and display the data. A similar tool, but with a somewhat different concept, has been described recently : Li XJ, Pedrioli PG, Eng J, Martin D, Yi EC, Lee H, Aebersold R., Anal Chem. 2004 Jul 1;76(13):3856-60. Comparing complex mixtures A B 1 1 3 3 2 2 582.?? 740.35( 2+) LGEYGFQNALIVR 627.6 3+, RPCFSALTPDETYVPK 631.7 3+, ?? Image alignment and superposition with two-colour display Landmarks were used to align and superimpose the maps to account for small differences in elution profiles Mix A: NO BSA added Mix B: A + 26 fmol BSA tryptic digest added +4 ion 5001000m/z Rationale : complex mixtures resulting from proteolysis of hundreds of proteins require multi-dimensional separations followed by dozens of LC-MS/MS runs to be efficiently sampled. Often most of the information produced is redundant since only those peptides that are found to vary between two samples are biologically meaningful. We have explored the feasibility of comparing complex peptide mixtures with our software tool after LC-MS analysis. A regular LC-MS/MS analysis of mix B identified BSA by matching 3 peptides About 30 BSA peptides were found by comparing mix A and B with the mapping tool Highly reproducible datasets can be generated if samples are run within the same measurement session Comparisons are possible and allow to highlight minor differences Figure 2 : comparing two peptide mixtures with a known added components A fraction corresponding to the protein mw range 26-33 kDa was isolated from a lysate of the human BJAB cell line and digested with trypsin. In a separate study, this sample was found to contain more than 180 proteins at detectable levels by LC-MS/MS. We compared the map we obtained for this sample with the same generated after addition of varying amounts of a standard protein digest. LC : nano- RP-HPLC (LC-Packings), 30 min gradient. MS : SCIEX QSTARi quadrupole-time-of flight. In-vivo PTM’s : a first look at a Histone fraction Figure 6 : mapping some easy-to-find PTM’s An histone-containing fraction was purified by acidic extraction of chromatin and subsequently digested with trypsin. Representation with the software tool and annotation of the map with MASCOT results helps locate some known post-translational modifications such as the N-terminal acetylation (  m=42.0, 21.0 for 2+ species) of linker Histones H1.2 and H1.5. The RT shift induced by acetylation shows a reproducible pattern. Other possibly modified ion pairs were identified which could represent differentially methylated peptides. Work in progress….  m=21 Da Figure 6 : mapping some high stoichiometry Histone PTM’s Conclusion The tool greatly facilitates the following tasks : overall data inspection and search for peptides of interest assessment of efficiency of sampling by MS/MS alignment and comparisons of two complex mixtures mapping of some known PTMs 2 Figure 3 : Artifacts 1 : multiple oxidations and metal adducts. Affected peptides contain Met, Asp and/or Glu. Figure 4 : Artifacts 2: more oxidations…residue affected unknown, possibly cysteine ? Peptide modifications due to sample treatment A region of the LC-MS map of a 2D-PAGE spot corresponding to RLA0_HUMAN (60S acidic ribosomal protein P0). In addition to mapping MASCOT-identified peptides we looked for strong peaks that were analysed by MS/MS but not database-matched, a task that is greatly simplified by the visualisation tool. The ion with m/z= 734.95 (2+) had been analysed by MS/MS but not matched to anything. Manual analysis of the CID spectrum showed it is a modified form of peptide IIQLLDDYPK (normally 609 2+), with the tyrosine residue displaying a mass of 414.0 Da ! The nature of this modification is under investigation. Figure 5 : Unexpected modifications : verifying the identity of prominent unmatched peptides 3 4 Introduction : the tool Perspectives More functionalities need to be developed to make this software tool even more powerful, namely : accurate peak detection and charge state determination locally flexible image alignment full annotation from MS/MS identification results scanning for peaks with mass differences corresponding to PTM’s The real power of the tool is in allowing differential display of two or more datasets. This requires careful experimental design to obtain comparable samples. raw data are displayed (no information is lost !) variable resolution display with zoom-in of more than a factor 1000x possible display of individual MS spectra, calculation of mass differences between peaks 5 6 582.35 (2+) LVNELTEFAK A B How complete is the sampling of a complex mixture ? 1 Figure 1: nanoLC-MS/MS analysis of a trypsinised fraction of nuclear proteins. Only a part of the whole chromatogram is displayed. Ions selected for MS/MS were annotated on the image and are labeled with blue crosses. Only a subset of these has been successfully matched to database sequences. The software tool can be successfully used to display annotated maps immediately giving an overview of the sampling efficiency of the MS/MS analysis and allowing to find out if peaks of interest have been analysed or not. landmark-based image alignment (warping only of the time dimension) two-colour display automatic annotation of images with MS/MS data any MS data types (high- and low-resolution) can be displayed