Characterization of the Velos, an Enhanced LTQ Orbitrap, for Proteomics

Fast and efficient peptide sequencing is a cornerstone of modern pro- teomics. We have evaluated and characterized a new and improved version of the LTQ Orbitrap for proteomics experiments. The new instru- ment is different in three key aspects from the current instrument. It’s source has a significantly higher ion transmission, it encompasses a significantly faster scanning linear ion trap and it has much more efficient Higher Energy C-trap Dissociation (HCD). These features – in combination with predictive automatic gain control – and parallel detector acquisition enable short cycle times (2.8 seconds) for Top20 MS/MS acquisition in the LTQ in combination with high-resolution full scan MS detection in the orbitrap. The new instrument also present a more than ten-fold improve- ment in its HCD capabilities in terms of speed and sensitivity, which in combination with the new brighter ion source allow for routine Top10 MS/MS cycles. The HCD spectra provide ppm-mass accuracy on all fragments spanning the full-mass-range (no low mass cut-off). High- resolution HCD in combination with online HPLC separation allow us to identify and quantify more than one thousand proteins in single runs of a complex peptide mixture. As a consequence of the spectacular HCD performance it is now possible to analyze both precursor and fragment ions with high-mass accuracy on a chromatographic scale without sacri-ficing acquisition speed or sensitivity. We have analyzed a whole yeast proteome lysate by peptide IEF fractionation in combination with HCD sequencing on the Velos and compared the haploid with the diploid state using SILAC. From three replicate analyses of 24 IEF fractions we are able to identify and quantify almost four thousand proteins with very low false discovery rate. Reversible phosphorylation of proteins, carried out by kinases and phos- phatases, constitutes one of the most important regulatory mechanisms in eukaryotic cells. The various kinases, phosphatases and their substrates form a network that controls and processes the flow of information, from sensors via signaling relays to effector molecules. This network is of fundamental importance for the function and robustness of essentially all biological systems. Here, we describe a label-free, quantitative phosphoproteomics ap-proach to determine the in vivo relationship between 97 kinases, 28 phosphatases and their cellular substrates in S. cerevisiae . We systemat-ically detected and identified phosphopeptides that showed a significant and reproducible change in their abundance upon removal (or inactivation) of each of these kinases or phosphatases. In total, we identified over 7,000 regulated phosphorylation events on 1,213 substrate proteins, describing the first system-wide in vivo protein phosphorylation network of S. cer- evisiae . Our data indicate the extent of biological activity of each kinase and phosphatase under the tested condition, associates many of them with specific cellular functions, and allows correlation of regulated phos-phoproteins with yeast growth and morphological phenotypes. Overall, these results expand the systems-level understanding of phosphorylation-modulated signaling in yeast. Protein phosphorylation is a main regulatory switch in the cell, controlling processes such as cell division, growth, proliferation, differentiation and survival. This control is performed by intricate signaling networks, which are capable of altering protein activities and rapidly communicating mes-sages from different extracellular or internal cues to ultimately promote adequate cell readjustments. Numerous studies have addressed protein phosphorylation over the past decades, often on a single protein/pathway level. However, the global picture of signaling events can only be accom-plished from comprehensive studies, which are becoming attainable by mass spectrometry (MS)-based proteomics. The main difficulty in MS large-scale phosphorylation studies is the limitation in detecting phosphorylated species within complex sample mixtures due to their low abundance. However, the past five years have seen a steady improvement in phosphopeptide enrichment and MS data acquisition methods along with the development of computational tools for data analysis and validation, allowing us to routinely identify thousands of phosphorylation events from a single experiment. An overview of these efforts and the current status of technologies to profile the phosphopro-teome will be given. Furthermore, these strategies have been combined with stable isotope labeling for quantitative studies where two cell populations in different phosphorylation status are compared. One of the most challenging prob-lems in signal transduction is establishing kinase-substrate relationships. We have combined chemical genetics and large-scale quantitative phosphoproteomics to identify in vivo substrates of the master mitotic kinase Cdk1 and meiotic kinase Ime2 in the budding yeast Saccharomyces cerevisiae . Using this approach, we expanded the number of known bona fide substrates to (cid:2) 400 for each kinase, and pinpointed the precise sites of phosphorylation. We observed that substrates for Cdk1 and Ime2 vastly overlap; however the sites targeted and the linear motif preferred seem to differ, which provides an example on how different layers of regulation are assembled in complex systems. To explore the mechanisms and evolution of cell-cycle control, we ana- lyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccha- romyces cerevisiae . We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phos- phorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase signaling circuits.