Post-Translational Modifications
Phosphopeptide Enrichment Strategies
Liquid Chromatography-Mass Spectrometry
Data Processing, Validation, and Analysis
Phosphorylation of proteins is the most studied and best understood post-translational modification. However, the analysis of phosphoproteins is still one of the most challenging tasks in proteome research for a few reasons. Specifically, only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time. The phosphorylated sites on proteins might vary due to the fact that many phosphoproteins exist in several different phosphorylated forms. Finally, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, and in the complex biological mixtures minor phosphorylated sites might be difficult to identify.
Thus, the isolation of pure phosphoproteins or phosphopeptide samples is essential for successful identification of phosphorylated sites. The most frequently used methods in isolation and detection of phosphoproteins and -peptides are specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques. Here, we outline methods for enrichment of phosphorylated proteins and peptides and their identification using mass spectrometry-based techniques employed in our laboratory.
We effectively use anti-phosphotyrosine antibodies to immunoprecipitate, and therefore enrich low-abundant tyrosine phosphorylated proteins from the complex mixtures of proteins such as cell lysates. However, these antibodies are not very good at enriching for phosphopeptides. Also, there are no antibodies for enriching proteins that are phosphorylated on serine or threonine residues.
For such cases we employed alternative methods which base on the phosphopeptide enrichment from proteolytic digest using titanium oxide-based solid-phase material (Titansphere). In this approach, phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO2 column (GL Sciences, Tokyo, Japan) while nonphosphorylated peptides are washed out. Subsequently, phosphopeptides are desorbed from the TiO2 column under alkaline conditions, and analyzed by nanoflow LC- MS/MS.
In the case of complex biological samples, we utilize multi-dimensional fractionation techniques to reduce the complexity of peptide mixtures prior subjecting samples to mass a spectrometric analysis. In the first dimension, samples are fractionated by off line strong cation exchange chromatography (SCX) into 8-10 fractions. Each fraction enriched by TiO2 column and subjected to a further separation using reversed phase chromatography in the second dimension following by mass spectrometry.
We utilize LC-MS based approach for separating peptide digest to decrease the complexity of the sample. In this method, peptides are loaded on the reverse-phase C18 nanocolumn (PepMap100, 75 µm x15 mm, 5 µm particle size, 100 ?, Dionex) and then eluted into a high resolution mass spectrometer (LTQ-FT or LTQ Orbitrap, ThermoFisher Scientific Co.) equipped with the nanosource. In the mass spectrometer, the first scan are performed by the survey scan acquisition in the FT-ICR or Orbitrap analyzer (at R of 30,000-100,000) followed by MS2 of the five most intense peptide ions and MS3 of all ions showing the neutral loss of phosphoric acid (98 Da) from the precursor ion in the LTQ. Coupling nanoLC system to a mass spectrometer is valuable since it decreases the ion suppression effect observed in the case of phosphopeptides.
Acquired tandem mass spectra are typically searched against NCBI or Swiss-Prot protein databases using Mascot search engine (Matrix Science). The search criteria are required a full proteolytic enzyme specificity and two missed cleavages for digestion; carboxymethilation, Met oxidation, phosphorylations (STY) that are set as variable modifications; precursor ion mass tolerance of 8 ppm and fragment ion tolerance of 0.8 Da are required for all MS and MSx2 measurements respectively. In addition, we manually validated all spectra using the following acceptance criteria: all phosphor-Ser and phosphor-Thr peptides required to show a pronounced neutral loss-dependent MSx3 scan and extensive coverage of b- and/or y-ion series.
Contact Person:
Janna Kiselar, Ph.D. (janna.kiselar@case.edu)
Case Center for Proteomics and Bioinformatics
Case Western Reserve University
10900 Euclid Avenue, BRB 934
Cleveland, OH 44106
(phone) 216-368-0979
(fax) 216-368-6846