We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an "analog-sensitive" Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.

Total analysis time depends on the sample preparation, the simpler this can be made, the more samples can be analyzed in a reasonable time frame. To reduce the sample preparation time high data quality, and orthogonal separation methods can be used to evaluate product complexity. Differential ion mobility has been shown to effectively reduce the data complexity and to separate features of different sizes and mobility. Here, the interferences in the spectra were separated from the ADC of interest.

Monoclonal antibodies (mAb) are major target-oriented biotherapeutics that are used to treat an array of human diseases. Current therapeutic monoclonal antibodies are immunoglobulin G (IgG) 1 derivatives, typically produced in mammalian cell culture using Chinese hamster ovary (CHO) or other cells. Process-related impurities and other trace contaminants that co-purify during downstream processing must be characterized and monitored to control product quality, potency, and safety.

METHODS. We investigated the interaction between HuR and Cdk1 using co-IP and the effects of cdk1 on HuR distribution using specific Cdk1 inhibitor and siRNA for cdk1, cdk7, and cyclin B1. To assess the effect of HuR phosphorylation on its localization, a novel phospho- HuR(S202) antibody was developed and point mutant HuR(S202A) was made. Binding of HuR to 14-3-3 θ was determined by co-IP and specific siRNA was used to check the role of 14-3-3 θ in HuR translocation. To determine the biological consequences of HuR phosphorylation, target mRNA binding was assessed by IP-RNP and etoposide was used as apoptotic inducer.

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.

Despite advances in understanding pluripotency through traditional cell biology and gene expression profiling, the signaling networks responsible for maintenance of pluripotency and lineage-specific differentiation are poorly defined. To aid in an improved understanding of these networks at the systems level, we present procedures for the combined analysis of the total proteome and total phosphoproteome (termed (phospho)proteome) from human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and their differentiated derivatives. Because there has been considerable heterogeneity in the literature on the culture of pluripotent cells, we first briefly describe our feeder-free cell culture protocol. The focus, however, is on procedures necessary to generate large-scale (phospho)proteomic data from the cells. Human cells are described here, but the (phospho)proteomic procedures are broadly applicable. Detailed procedures are given for lysis of the cells, protein sample preparation and digestion, multidimensional liquid chromatography, analysis by tandem mass spectrometry, and database searches for peptide/protein identification (ID). We summarize additional data analysis procedures, the subject of ongoing efforts.

Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a 15N for a 13C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a 13C was exchanged for a 15N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the 13C- and 15N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex.

The considerable progress in high-throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the past decade has been fueled to a large degree by continuous improvements in instrumentation. High-throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times 2 times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker's yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 full width at half-maximum (fwhm), it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.

Introduction Comprehensive definition of a proteome is a necessary foundation for subsequent investigations of proteome dynamics. Proteome characterization should be as efficient, reliable, and exhaustive as possible. Tandem mass spectrometry, particularly using linear ion traps and trap-based hybrid mass spectrometers, has become the technology of choice for peptide and protein identification. The preference for this instrumentation is attributable to robustness, ease of use, superior MS and especially MS/MS performance. Undiscovered components within a proteome frequently include dynamically modified forms which exist at abundance levels undetectable by most instrumentation. Therefore, the development of more sophisticated instrumentation is essential for a more exhaustive characterization of complex proteomes. This application note addresses the challenging analysis of a complex, multi-organ peptide digest of Caenorhabditis elegans (C. elegans) to assess the performance of an innovative new dual-pressure linear ion trap mass spectrometer. Performance of the new instrument is benchmarked against the previous state-ofthe-art linear ion trap and also against a well-known quadrupole time-of-flight (Q-TOF) mass spectrometer. Instrument Innovations The instrument evaluated in this note is the Thermo Scientific LTQ Velos ion trap mass spectrometer. It includes a novel dual-pressure linear ion trap and a highpressure stacked ring ion guide (S-lens), as shown in Figure 1. The radio frequency (RF) S-lens significantly increases transmission of ions into the mass analyzer. This reduces the time required to inject the desired ion population into the linear ion trap. The first cell of the dual-pressure trap is held at a higher pressure (~5 x 10-3 Torr) than in previous linear ion trap systems to improve efficiency of trapping, isolating, and fragmenting ions of interest. The increased isolation efficiency produces a 4-fold decrease in the time required for precursor ion isolation. The geometry of the LTQ VelosTM ion trap allows better selectivity for lowabundance precursors in the presence of abundant interfering ions, improving dynamic range for MS/MS analysis. The higher pressure also reduces the required CID activation time by 67%, while maintaining the same efficiency of fragmentation. The second cell, held at a lower pressure (~4 x 10-4 Torr), allows for a faster mass analysis scan with increased resolution. In conjunction with the new dual-pressure linear ion Key Words

This study uses the challenging analysis of a complex, multi-organ peptide digest of Caenorhabditis elegans (C. elegans) to compare the performance of a novel linear ion trap mass spectrometer and a quadrupole time-of-flight (Q-TOF) mass spectrometer.

Elongation of telomeres by telomerase replenishes the loss of terminal telomeric DNA repeats during each cell cycle. In budding yeast, Cdc13 plays an essential role in telomere length homeostasis, partly through its interactions with both the telomerase complex and the competing Stn1-Ten1 complex. Previous studies in yeast have shown that telomere elongation by telomerase is cell cycle dependent, but the mechanism underlying this dependence is unclear. In S. cerevisiae, a single cyclin-dependent kinase Cdk1 (Cdc28) coordinates the serial events required for the cell division cycle, but no Cdk1 substrate has been identified among telomerase and telomere-associated factors. Here we show that Cdk1-dependent phosphorylation of Cdc13 is essential for efficient recruitment of the yeast telomerase complex to telomeres by favoring the interaction of Cdc13 with Est1 rather than the competing Stn1-Ten1 complex. These results provide a direct mechanistic link between coordination of telomere elongation and cell-cycle progression in vivo.

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.

AMPA receptors mediate fast excitatory synaptic transmission and are essential for synaptic plasticity. ANQX, a photoreactive AMPA receptor antagonist, is an important biological probe used to irreversibly inactivate AMPA receptors. Here, using X-ray crystallography and mass spectroscopy, we report that ANQX forms two major products in the presence of the GluR2 AMPAR ligand-binding core (S1S2J). Upon photostimulation, ANQX reacts intramolecularly to form FQX or intermolecularly to form a covalent adduct with Glu705.

We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-and-release methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo , but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase–substrate connections in signaling networks.

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.