Abstract
In proteomics multi-dimensional fractionation techniques are widely used to reduce the complexity of peptide mixtures subjected to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in the separation of peptides generated by a relatively little explored metallo-endopeptidase with Lys-N cleavage specificity. When such proteolytic peptides are subjected to low-pH strong cation exchange we obtain fractionation profiles in which peptides from different functional categories are well separated. The four categories we distinguish and are able to separate to near completion are (I) acetylated N-terminal peptides; (II) singly phosphorylated peptides containing a single basic (Lys) residue; (III) peptides containing a single basic (Lys) residue; and (IV) peptides containing more than one basic residue. Analyzing these peptides by LC-MS/MS using an ion trap with both collision as well as electron transfer-induced dissociation provides unique optimal targeted strategies for proteome analysis. The acetylated peptides in category I can be identified confidently by both CID and ETcaD, whereby the ETcaD spectra are dominated by sequence informative Z-ion series. For the phosphorylated peptides in category II and the "normal" single Lys containing peptides in category III ETcaD provides unique straightforward sequence ladders of c'-ions, from which the exact location of possible phosphorylation sites can be easily determined. The later fractions, category IV, require analysis by both ETcaD and CID, where it is shown that electron transfer dissociation performs relatively well for these multiple basic residues containing peptides, as is expected. We argue that the well resolved separation of functional categories of peptides observed is characteristic for Lys-N-generated peptides. Overall, the combination of Lys-N proteolysis, low-pH strong cation exchange, and reversed phase separation, with CID and ETD induced fragmentation, adds a new very powerful method to the toolbox of proteomic analyses.
Highlights
In proteomics multi-dimensional fractionation techniques are widely used to reduce the complexity of peptide mixtures subjected to mass spectrometric analysis
We developed a method for global protein analysis of whole cell lysates using a combination of Lys-N proteolytic cleavage followed by low-pH strong cation exchange (SCX) fractionation and reversed phase (RP)-nanoflow liquid chromatography (nanoLC)-ETcaD-Mass spectrometry (MS) analysis and RP-nanoLC-CID-MS analysis
We explored a method for global protein analysis of whole cell lysates using a combination of Lys-N proteolytic cleavage followed by low-pH SCX fractionation and RP-nanoLCMS/MS analysis using both CID and ETcaD
Summary
Materials—Protease inhibitor mixture was obtained from Roche Diagnostics. Metallo-endopeptidase from Grifola Frondosa (Lys-N) was obtained from Seikagaku Corporation (Tokyo, Japan). ETD Experiments—The 49 dried SCX fractions were diluted in 100 l of 10% formic acid, and 1/20 (5 l) of all the SCX fractions were subjected to nanoscale liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis, performed on an Agilent 1100 HPLC system (Agilent technologies) connected to a LTQ XL Linear Ion Trap Mass Spectrometer with an ETD source at the back from Thermo Fisher Scientific, Inc. Spectra were searched against the International Protein Index Human database version 3.36 (69012 sequences; 29002682 residues) using Mascot software version 2.2.0, with Lys-N cleavage specificity. Tandem mass spectra assigned with a Mascot Score Ն30 (p value Յ0.05) were accepted providing a false discovery rate for the CID data of 4.68% and 2.53% for ETD, determined using a decoy database. For ETD spectra, exceptions occurred when c- or z-type ions (or related ions with ammonia/water losses) were assigned to the same isotope cluster in which case the most appropriate assignment (e.g. based on mass accuracy trend, mono isotopic peak etc) was chosen in an automated fashion
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