Peptide sequence analysis using a combination of gas-phase ion͞ion chemistry and tandem mass spectrometry (MS͞MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion͞ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS͞MS spectra of phosphopeptides separated by nanoflow HPLC is described.electron capture dissociation ͉ fragmentation ͉ ion͞ion reactions ͉ charge transfer ͉ ion trap S ix years ago, McLafferty and coworkers (1) introduced a unique method for peptide͞protein ion fragmentation: electron capture dissociation (ECD). In this method, multiply protonated peptides or proteins are confined in the Penning trap of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and exposed to electrons with near-thermal energies. Capture of a thermal electron by a protonated peptide is exothermic by Ϸ6 eV (1 eV ϭ 1.602 ϫ 10 Ϫ19 J) and causes the peptide backbone to fragment by a nonergodic process, e.g., one that does not involve intramolecular vibrational energy redistribution (2-5). One pathway for this process involves generation of an odd-electron hypervalent species (RNH 3 • ) that dissociates to produce RNH 2 and a hydrogen radical (6). As shown in Fig to an amide nitrogen, a secondary pathway, leads to the formation of carbon monoxide plus a homologous series of complementary fragment ions of types a and y. Subtraction of the m͞z values for the fragments within a given ion series that differ by a single amino acid affords the mass and thus the identity of the extra residue in the larger of the two fragments. The complete amino acid sequence of a peptide is deduced by extending this process to all homologous pairs of fragments within a particular ion series.Because ECD occurs along the peptide backbone in a sequence-independent manner, preserves posttranslational modifications (PTMs) (7-14), and can be implemented on a millisecond time scale with precursor-to-product ion conversion efficiencies that approach 30% (15-21), it has become the technique of choice for the analysis of peptide and proteins with FTICR mass spectrometers (22-28). Unfortunately, ECD in its most efficient form requires that the precursor sample ions be immersed in a dense population of near-thermal electrons. Emulating these conditions in the instruments used most commonly for peptide and protein analyses, those that trap ions with radio frequency (RF) electrostatic fields rather than with static magnetic and electric fields, remains technically challenging. Thermal electrons introduced into the RF fields of RF 3D quadrupole ion trap (QIT), quadrupole time-of-flight, or RF linear 2D quadrupole ion trap (QLT) instruments maintain their thermal energy only for a fraction of a micros...

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSHbeads or anti-Myc linked to magnetic beads MK21 mediates several p38␣,␤ MAP kinase-dependent processes (for a review, see Ref. 1), demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2). MK2 (Ϫ,Ϫ) mice have suppressed stress responses. Cellular studies show deficits in motility, chemotaxis, and cytokine production. Macrophages taken from MK2 (Ϫ,Ϫ) mice exhibit normal TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF protein was markedly decreased in MK2 (Ϫ,Ϫ) macrophages, suggesting a block in production of TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and translation (3, 4) but is not completely understood.The p38␣,␤ MAP kinase pathway regulates stability of mRNAs that contain AU-rich elements in their 3Ј-untranslated regions. Examples include TNF, COX-2, interleukin-6, and interleukin-1␤ (4 -6). Evidence chiefly comes from mRNA stabilization caused by transfection of mutationally activated MEK3/MEK6 or by the addition of agents that activate p38 MAPK and conversely from destabilization caused by the addition of a p38␣,␤ MAP kinase inhibitor. Since p38␣,␤ is required to activate MK2, these experiments do not dissect contributions from MK2. Studies in MK2 (Ϫ,Ϫ) cells suggest that MK2 regulates stability of some cytokine mRNAs (2, 4). Lasa et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2 blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine production post-transcriptionally is unknown. Mahtani et al. (7) reported that tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.TTP (for a review, see Ref. 8) destabilizes class II AU-rich elements and is the prototype for a non-zinc finger class of nucleic acid-binding proteins. Destabilization requires integrity of the TTP tandem Cys 3 His RNA binding domains that coordinate zinc in a disklike structure (9 -11). TTP-null mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-like symptoms attributed to increased production of TNF (12).TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation occurs at more than one site evident by the appearance of two distinct slower migrating forms of TTP on gels after stimulation that are reversed by phosphatase treatment (7, 13). TTP under...

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions ofa lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

PurposeWe examined associations between tumor characteristics (human epidermal growth factor receptor 2 [HER2] protein expression, HER2 gene and chromosome 17 copy number, hormone receptor status) and disease-free survival (DFS) of patients in the N9831 adjuvant trastuzumab trial.Patients and MethodsAll patients (N = 1,888) underwent chemotherapy with doxorubicin and cyclophosphamide, followed by weekly paclitaxel with or without concurrent trastuzumab. HER2 status was determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) at a central laboratory, Mayo Clinic, Rochester, MN. Patients with conflicting local positive HER2 expression results but normal central laboratory testing were included in the analyses (n = 103).ResultsPatients with HER2-positive tumors (IHC 3+, FISH HER2/centromere 17 ratio ≥ 2.0, or both) benefited from trastuzumab, with hazard ratios (HRs) of 0.46, 0.49, and 0.45, respectively (all P < .0001). Patients with HER2-amplified tumors with polysomic (p17) or normal (n17) chromosome 17 copy number also benefited from trastuzumab, with HRs of 0.52 and 0.37, respectively (P < .006). Patients who received chemotherapy alone and had HER2-amplified and p17 tumors had a longer DFS than those who had n17 (78% v 68%; P = .04), irrespective of hormone receptor status or tumor grade. Patients with HER2-normal tumors by central testing (n = 103) seemed to benefit from trastuzumab, but the difference was not statistically significant (HR, 0.51; P = .14). Patients with hormone receptor–positive or –negative tumors benefited from the addition of trastuzumab, with HRs of 0.42 (P = .005) and 0.60 (P = .0001), respectively.ConclusionThese results confirm that IHC or FISH HER2 testing is appropriate for patient selection for adjuvant trastuzumab therapy. Trastuzumab benefit seemed independent of HER2/centromere 17 ratio and chromosome 17 copy number.

High school soccer injury patterns vary by gender and type of exposure. Identifying such differences in injury patterns is the important first step in the development of evidence-based, targeted injury prevention efforts.

The Epic Platform was developed for the unbiased detection and molecular characterization of circulating tumour cells (CTCs). Here, we report assay performance data, including accuracy, linearity, specificity and intra/inter-assay precision of CTC enumeration in healthy donor (HD) blood samples spiked with varying concentrations of cancer cell line controls (CLCs). Additionally, we demonstrate clinical feasibility for CTC detection in a small cohort of metastatic castrate-resistant prostate cancer (mCRPC) patients. The Epic Platform demonstrated accuracy, linearity and sensitivity for the enumeration of all CLC concentrations tested. Furthermore, we established the precision between multiple operators and slide staining batches and assay specificity showing zero CTCs detected in 18 healthy donor samples. In a clinical feasibility study, at least one traditional CTC/mL (CK+, CD45-, and intact nuclei) was detected in 89 % of 44 mCRPC samples, whereas 100 % of samples had CTCs enumerated if additional CTC subpopulations (CK-/CD45- and CK+ apoptotic CTCs) were included in the analysis. In addition to presenting Epic Platform's performance with respect to CTC enumeration, we provide examples of its integrated downstream capabilities, including protein biomarker expression and downstream genomic analyses at single cell resolution.

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