Thomas Aarup Hansen

Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO(2) Chromatography Applied to Proximal EGF Signaling

Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.

Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones.

We present an integrated middle‐down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5–7 kDa). We combined an RP trap column with subsequent weak cation exchange–hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N‐terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12−/−) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven‐fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12−/− cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.

Reduction in Database Search Space by Utilization of Amino Acid Composition Information from Electron Transfer Dissociation and Higher-Energy Collisional Dissociation Mass Spectra

With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

Deconvolution of mixture spectra and increased throughput of peptide identification by utilization of intensified complementary ions formed in tandem mass spectrometry.

A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high-resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial data set.

Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry.

Reversible protein phosphorylation plays a critical role in cell signaling and is responsible for the regulation of many biological processes in most living organisms. The low stoichiometry of protein phosphorylation requires sensitive analysis by tandem mass spectrometry. However, incomplete peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho-site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ∼32 compared to ∼23 and ∼17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD), respectively. Compared with the Ascore using either CAD or ETD, the Cscore identified up to 88% more phosphorylation sites. Using a phosphopeptide library revealed that the score threshold for obtaining a false-localization rate of 0.5% was lower for the Cscore than either the Ascore (CAD) or the Ascore (ETD).

Electron Transfer Dissociation Reveals Changes in the Cleavage Frequencies of Backbone Bonds Distant to Amide-to-Ester Substitutions in Polypeptides

Interrogation of electron transfer dissociation (ETD) mass spectra of peptide amide-to-ester backbone bond substituted analogues (depsipeptides) reveals substantial differences in the entire backbone cleavage frequencies. It is suggested that the point permutation of backbone bonds leads to changes in the predominant ion structures by removal/weakening of specific hydrogen bonding. ETD responds to these changes by redistributing the cleavage frequencies of the peptide backbone bonds. In comparison, no distinction between depsi-/peptide was observed using collision-activated dissociation, which is consistent with a general unfolding and elimination of structural information of these ions. These results should encourage further exploration of depsipeptides for gas-phase structural characterization.