Improvement of quality of peptide mass spectra in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay analysis of salty protein digests by using on-target washing † Proteomics usually uses a suitable technique for separation of proteins that are then subjected to digestion with an enzyme and subsequently the peptides obtained are measured with a mass spectrometric technique, e.g. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). 1 Experimentally measured peptide masses are compared with theoretical masses of all possible enzyme-derived peptides for each protein included in protein databases and the protein is identified on the basis of the best agreement of all such comparisons. Peptide mass fingerprinting (PMF) 2 -6 is a sensitive and rapid method with one practical advantage: only part of the sample (about 10%) is consumed for the analysis. In the case when PMF fails, the other part of the sample can be analyzed by tandem mass spectrometry (e.g. ion-trap, triple quadrupole, quadrupole-TOF, TOF-TOF). Another possibility is post-source decay (PSD) analysis 7,8 using a MALDI-TOF instrument equipped with an ion gate and a reflectron. This approach is sensitive and no further sample handling steps are necessary, as the sample is already prepared from the PMF experiment.
The sample preparation in MALDI-TOFMS is a crucial step for successful protein identification using both PMF and PSD analysis. The samples are often contaminated with salts that are necessary for successful separation or enzymatic digestion of the investigated protein. The presence of salts can suppress the peptide signals in MALDI-TOFMS, which is why an efficient desalting procedure must be employed. One possibility is to use a reversed-phase microcolumn (e.g. ZipTip pipette tips) for desalting and preconcentration of the peptide sample. 9 Also, the use of prestructured MALDI-TOFMS sample supports is often used for improving the MALDI signal of contaminated samples. 10 -12 However, the cheapest and easiest way to remove salts from the contaminated sample is to wash the deposited sample directly on the target before MALDI-TOFMS measurements. Simple immersion of the probe tip with the drieddroplet deposited sample in cold water for 15 s was employed. 13 The deposition of a large droplet (about 5 µl) of deionized water or dilute organic acid (trifluoroacetic or formic acid) on the deposited sample for a few seconds (2-10 s) with subsequent removing of the droplet was also successfully used for improving MALDI-TOF mass spectra. 1,14 -16 The washing procedure can be repeated several times provided that the matrix film is not damaged. 1 In this work, we used size-exclusion chromatography (SEC) for protein separation and MALDI-TOFMS for the characterization of peptides derived from in-solution digested proteins. The aim was to apply and optimize the on-target washing procedure for MALDI-TOFMS measurements and PSD analysis of peptide samples with high salt content. The number of washing steps and the time of the washing step were optimized. For monitoring of the sample losses during the washing, the analysis after washing of the collected droplet was carried out. The improvement of the signal in MALDI-TOFMS measurements is shown also in the PSD analysis of a tryptic peptide from horse heart transferrin.
The peptide in-solution digest of transferrin from horse heart (Sigma-Aldrich, Schnelldorf, Germany; SwissProt Q29443, 77 753 Da) containing the chromatographic mobile phase was used for this study. The fraction of transferrin in phosphate buffer was collected from SEC run at a concentration of transferrin of ¾5 pmol µl 1 .