38 research outputs found
Characterization of Microorganisms of Interest to Homeland Security and Public Health Utilizing Liquid Chromatography/Mass Spectrometry
Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Automated charge state deconvolution, spectral subtraction and spectral mirroring were used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarkers were discovered based on their unique mass, retention time and relative intensity. These markers were implemented to differentiate closely related strain types, (e.g. two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. Notable, was the distinction of multiple serotypes of enterohaemorrhagic E. coli which cannot be distinguished by clinical manifestation alone. Additionally, speciation of Shigella was achieved, a task for which no commercial real-time polymerase chain reaction (PCR) primers exist. This method was subsequently applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Due to the increased difficulty during lysis, two new lysis protocols were developed, and each extracted a distinct set of proteins (by both mass and retention time). Extracts from both lysis procedures were utilized to discover biomarkers useful for identification and characterization at the species and strain levels. These biomarkers were successfully implemented to identify unknowns during a blind study and would enhance serological and genetic approaches by serving as new targets for detection. Two sets of the C. perfringens isolates that were deemed 100% similar by the gold standard for strain differentiation, pulsed-field gel electrophoresis (PFGE), were distinguished using LC/MS, demonstrating the high specificity of this approach. The final part of this work demonstrated the application of ultra performance liquid chromatography (UPLC) to this project to improve the throughput of the method. Given that numerous small molecule applications of UPLC have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of ten protein standards, the optimized UPLC method yielded improved chromatographic resolution, enhanced sensitivity, and a three-fold increase in throughput. Application of this method to cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality; a reduction in run time from 75 minutes to 25 minutes was achieved
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Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.
The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites-the global pattern of phosphorylation or 'phospho-form'-giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2(n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods-western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)-on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem
Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine
Selenoproteins are a unique group of proteins that contain selenium in the
form of selenocysteine (Sec) co-translationally inserted in response to a UGA
codon with the help of cis- and trans-acting factors. Mammalian selenoproteins
contain single Sec residues, with the exception of selenoprotein P (SelP) that
has 7–15 Sec residues depending on species. Assessing an individual’s selenium
status is important under various pathological conditions, which requires a
reliable selenium biomarker. Due to a key role in organismal selenium
homeostasis, high Sec content, regulation by dietary selenium, and
availability of robust assays in human plasma, SelP has emerged as a major
biomarker of selenium status. Here, we found that Cys is present in various
Sec positions in human SelP. Treatment of cells expressing SelP with
thiophosphate, an analog of the selenium donor for Sec synthesis, led to a
nearly complete replacement of Sec with Cys, whereas supplementation of cells
with selenium supported Sec insertion. SelP isolated directly from human
plasma had up to 8% Cys inserted in place of Sec, depending on the Sec
position. These findings suggest that a change in selenium status may be
reflected in both SelP concentration and its Sec content, and that
availability of the SelP-derived selenium for selenoprotein synthesis may be
overestimated under conditions of low selenium status due to replacement of
Sec with Cys
Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P
Selenoprotein biosynthesis relies on the co-translational insertion of
selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere
with ribosomal function and may cause codon misreading. We hypothesized that
biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is
particularly sensitive to antibiotics due to its ten in frame UGA codons. As
liver regulates Se metabolism, we tested the aminoglycosides G418 and
gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in
experimental mice. In vitro, SELENOP levels increased strongly in response to
G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was
marginally affected. Se content of G418-induced SELENOP was dependent on Se
availability, and was completely suppressed by G418 under Se-poor conditions.
Selenocysteine residues were replaced mainly by cysteine, tryptophan and
arginine in a codon-specific manner. Interestingly, in young healthy mice,
antibiotic treatment failed to affect Selenop biosynthesis to a detectable
degree. These findings suggest that the interfering activity of
aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the
Se status, and other parameters likely including age and general health.
Focused analyses with aminoglycoside-treated patients are needed next to
evaluate a possible interference of selenoprotein biosynthesis by the
antibiotics and elucidate potential side effects
Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer
As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h
Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins
Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDPglucose: glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins
Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans
Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. We used Tandem Mass Tag-labeled quantitative proteomics detecting 2323 proteins in a cohort comprising patients with DILI [at onset (DO) and follow-up (DF)], acute non-DILI [at onset (NDO) and follow-up (NDF)], and healthy volunteers (HV) to identify novel serum biomarkers. Thirteen candidates selected based on differential expression, liver-specific expression, and mechanistic relevance to liver pathology, were assessed in confirmatory and replication cohorts of HV (n=94), DO (n=123), DF (n=110), NDO (n=58) and NDF (n=37) using a targeted label-free SureQuant assay. Area under the receiver operating characteristic curve (AUC) ranging between 0.94 and 0.99 across cohorts for five of these biomarkers, reflected differentiation between DO and HV with high sensitivity and specificity. In addition, fructose-1,6-bisphosphatase 1 distinguished NDO from DO (AUC: 0.75 and 0.65) on its own or in combination with glutathione S-transferase A1 and leukocyte cell derived chemotaxin 2 (AUC: 0.78 and 0.68). These can potentially differentiate DILI and acute liver injury from non-drug etiologies
MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments
Protein
phosphorylation is critically important for many cellular
processes, including progression through the cell cycle, cellular
metabolism, and differentiation. Isobaric labeling, for example, tandem
mass tags (TMT), in phosphoproteomics workflows enables both relative
and absolute quantitation of these phosphorylation events. Traditional
TMT workflows identify peptides using fragment ions at the MS2 level
and quantify reporter ions at the MS3 level. However, in addition
to the TMT reporter ions, MS3 spectra also include fragment ions that
can be used to identify peptides. Here we describe using MS3 spectra
for both phosphopeptide identification and quantification, a process
that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize
several instrument parameters, including the modality of mass analyzer
(i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and
MS3 normalized collision energy (NCE), to achieve the best balance
of identified and quantified peptides. Our optimized MS3-IDQ method
included the following parameters for the MS3 scan: NCE = 37.5 and
AGC target = 1.5 × 10<sup>5</sup>, and scan range = 100–2000.
Data from the MS3 scan were complementary to those of the MS2 scan,
and the combination of these scans can increase phosphoproteome coverage
by >50%, thereby yielding a greater number of quantified and accurately
localized phosphopeptides