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Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.

PURPOSE: To evaluate cellular protein changes in response to treatment with an approved drug, ibrutinib, in cells expressing normal or mutated Granulocyte-Colony Stimulating Factor Receptor (G-CSFR). G-CSFR mutations are associated with some hematological malignancies. Previous studies showed the efficacy of ibrutinib (a Bruton’s tyrosine kinase inhibitor) in mutated G-CSFR leukemia models but did not addressed broader signaling mechanisms. EXPERIMENTAL DESIGN: A label-free quantitative proteomics workflow to evaluate the cellular effects of ibrutinib treatment was established. This included 3 biological replicates of normal and mutated G-CSFR expressed in a mouse progenitor cell (32D cell line) with and without ibrutinib treatment. RESULTS: The proteomics dataset showed about 1,000 unique proteins quantified with nearly 400 significant changes (p value < 0.05), suggesting a highly dynamic network of cellular signaling in response to ibrutinib. Importantly, the dataset was very robust with coefficients of variation (CVs) for quantitation at 13.0-20.4% resulting in dramatic patterns of protein differences among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: This robust data set is available for further mining, hypothesis generation, and testing. A detailed understanding of the restructuring of the proteomics signaling cascades by ibrutinib in leukemia biology will provide new avenues to explore its use for other related malignancies.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8 ± 0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

SynopsisObjective The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. Methods The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. Results In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. Conclusion This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS.

Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.

A proteomic analysis was performed comparing normal rat soleus muscle to denervated soleus muscle at 0.5, 1, 2, 4, 6, 8 and 10 days post denervation. Muscle mass measurements demonstrated that the times of major mass changes occurred between 2 and 4 days post denervation. Proteomic analysis of the denervated soleus muscle during the atrophy process demonstrated statistically significant (at the p < 0.01 level) changes in 73 soleus proteins, including coordinated changes in select groups of proteins. Sequence analysis of ten differentially regulated proteins identified metabolic proteins, chaperone and contractile apparatus proteins. Together these data indicate that coordinated temporally regulated changes in the proteome occur during denervation-induced soleus muscle atrophy, including changes in muscle metabolism and contractile apparatus proteins.

Gerzrldk& K C I I I W ~ ~ 11. Greis, I,.-Y. Dermis I h g , Melissa A. Bloinherg Teh-Yirig Chou, MatiSliiow Jiatg Llizaheth I? I ~ ~ I ~ I I I ~ ~ c , loris M. Snow, Lisa K. Kreppel, Itoberl C. Cole atid Bradley K. Hayes. Dept. Biocheni. & Molecular Genetics, University of Alabama at Birmingham, UAB Station, 1918 University Avenue, Birmingham, AL 35294-0005;Phone: (205) 934-4753; FAX: (205) 975-6685; Internet: GWHART@BMG.BHS.UAB.EDU.