Despite advances in genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein level information. Formalin-fixed paraffin-embedded (FFPE) tissue specimens with extended clinical outcomes are widely available. Here, we perform comprehensive proteomic profiling of 300 FFPE breast cancer surgical specimens, 75 of each PAM50 subtype, from patients diagnosed in 2008-2013 (n = 178) and 1986-1992 (n = 122) with linked clinical outcomes. These two cohorts are analyzed separately, and we quantify 4214 proteins across all 300 samples. Within the aggressive PAM50-classified basal-like cases, proteomic profiling reveals two groups with one having characteristic immune hot expression features and highly favorable survival. Her2-Enriched cases separate into heterogeneous groups differing by extracellular matrix, lipid metabolism, and immune-response features. Within 88 triple-negative breast cancers, four proteomic clusters display features of basal-immune hot, basal-immune cold, mesenchymal, and luminal with disparate survival outcomes. Our proteomic analysis characterizes the heterogeneity of breast cancer in a clinically-applicable manner, identifies potential biomarkers and therapeutic targets, and provides a resource for clinical breast cancer classification.
Highlights d 45% of colon cancers sub-stochiometrically lose m 1 acp 3 J ribosomal RNA modification d 22+ distinct cancer types show hypo-modification of m 1 acp 3 J d The >1-billion-years-conserved m 1 acp 3 J is involved in ribosomal P site stability d Loss of m 1 acp 3 J modification drives heterogenous translation of RP mRNAs
Low-temperature plasma ionization, a technique that causes minimal fragmentation during ionization, is investigated as an ionization technique for mass spectrometric detection of the compounds in ambient organic aerosols in real time. The experiments presented in this paper demonstrate that ions are generated from compounds in the aerosol particles. The utility of this technique for detection of both positive and negative ions from the pyrolysate of multiple natural polymers is presented. Ultimately, low-temperature plasma ionization is shown to be a promising ionization technique for detection of compounds in organic aerosols by mass spectrometry.
Optimizing
the quality of proteomics data collected from a mass
spectrometer (MS) requires careful selection of acquisition parameters
and proper assessment of instrument performance. Software tools capable
of extracting a broad set of information from raw files, including
meta, scan, quantification, and identification data, are needed to
provide guidance for MS system management. In this work, direct extraction
and utilization of these data is demonstrated using RawTools, a standalone
tool for extracting meta and scan data directly from raw MS files
generated on Thermo Orbitrap instruments. RawTools generates summarized
and detailed plain text outputs after parsing individual raw files,
including scan rates and durations, duty cycle characteristics, precursor
and reporter ion quantification, and chromatography performance. RawTools
also contains a diagnostic module that includes an optional “preview”
database search for facilitating informed decision-making related
to optimization of MS performance based on a variety of metrics. RawTools
has been developed in C# and utilizes the Thermo RawFileReader library
and thus can process raw MS files with high speed and high efficiency
on all major operating systems (Windows, MacOS, Linux). To demonstrate
the utility of RawTools, the extraction of meta and scan data from
both individual and large collections of raw MS files was carried
out to identify problematic characteristics of instrument performance.
Taken together, the combined rich feature-set of RawTools with the
capability for interrogation of MS and experiment performance makes
this software a valuable tool for proteomics researchers.
Erythrocyte lysis and fish mortality assays, in combination with high performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis, were investigated for bioassay-guided fractionation of cultured golden alga (Prymnesium parvum). Intracellular constituents from isolated cell pellets and extracellular supernatant growth medium were fractionated by a variety of common separation modes, including reversed phase and normal phase solid phase extraction step fractionation procedures. For reversed phase fractionation of extracellular growth medium, one fraction was obtained that displayed hemolytic activity and adversely affected fish survival. Effective dose concentrations for this sample were similar in both assays and the LC-ESI-MS analysis of the fraction showed a number of mass spectral signals which were distinct to this fraction. Fractions obtained from separation of an ethanol extract of the lyophilized cell pellet provided one sample that was highly hemolytic, but not toxic to fish. Discrepancies such as this, along with notable fish behavioral responses from other nonhemolytic cell pellet fractions, problems with the use of unbonded silica gel for fractionation, and misleading mass spectral signatures are interesting in the context of our current understanding of P. parvum toxicity and remain to be investigated further. This work provides an account of ongoing research aimed toward comprehensive elucidation of toxic constituents produced by golden alga for the purpose of providing a better understanding and means to potentially remediate the ecological impact of this harmful bloom organism.(KEY TERMS: erythrocyte lysis; fish mortality; liquid chromatography; electrospray ionization; mass spectrometry; prymnesin.)
Extractive electrospray ionization is an ambient ionization technique that allows real-time sampling of liquid samples, including organic aerosols. Similar to electrospray ionization, the composition of the electrospray solvent used in extractive electrospray ionization can easily be altered to form metal cationized molecules during ionization simply by adding a metal salt to the electrospray solvent. An increase in sensitivity is observed for some molecules that are lithium, sodium, or silver cationized compared with the protonated molecule formed in extractive electrospray ionization with an acid additive. Tandem mass spectrometry of metal cationized molecules can also significantly improve the ability to identify a compound. Tandem mass spectrometry of lithium and silver cationized molecules can result in an increase in the number and uniqueness of dissociation pathways relative to [M + H]. These results highlight the potential for extractive electrospray ionization with metal cationization in analyzing complex aerosol mixtures. Graphical Abstract ᅟ.
The negative ion mode ESI mass spectral analysis of antimony(III)-D-and -L-tartrate ("tartar emetic"), in association with leucine enantiomeric isotopomers, revealed remarkable protonassisted enantioselective molecular recognition phenomena. The current study infers that recognition of amino acids by antimony(III)-D,L-tartrate complexes requires that the chiral selector associate a proton to become enantioselective. The dianionic selector itself failed to show enantiomeric discrimination capacity. This observation was shown to be consistent both in solution-phase targeting full scan and gas-phase targeting collision threshold dissociation (CTD) experiments. Importantly, this disparity in enantioselective binding capacity between the dianionic and the protonated monoanionic representatives of antimony(III)-D-and -L-tartrates could only be clearly revealed by ESI-MS and tandem mass spectrometry experiments as described herein. This finding urges a more in-depth study of mechanisms associated with exhibited enantiomeric resolving capacity of antimony tartrates in HPLC and CE applications, as well as in former ESI-MS association studies. (J Am Soc Mass Spectrom 2009, 20, 2100 -2105
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