38 research outputs found
Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry
Unravelling of disulfide linkage
patterns is a crucial part of
protein characterization, whether it is for a previously uncharacterized
protein in basic research or a recombinant pharmaceutical protein.
In the biopharmaceutical industry, elucidation of the cysteine connectivities
is a necessity to avoid disulfide scrambled and incorrectly folded
forms in the final product. Mass spectrometry (MS) is a highly utilized
analytical tool for this due to fast and accurate characterization.
However, disulfide bonds being an additional covalent bond in the
protein structure represent a challenge in protein sequencing by tandem
MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has
recently been demonstrated to provide efficient reduction efficiencies,
significantly enhancing sequence coverages in online coupling with
MS characterization. In this study, the potential use of EC disulfide
reduction in combination with MS characterization for disulfide mapping
was assessed. We employed two approaches based on (1) the high flexibility
and instant information about the degree of reduction in infusion
EC-MS to generate partially reduced species on the intact protein
level and (2) the preserved link between parent disulfide-linked fragments
and free reduced peptides in an LC–EC-MS platform of nonreduced
proteolytic protein digestions. Here we report the successful use
of EC as a partial reduction approach in mapping of disulfide bonds
of intact human insulin (HI) and lysozyme. In addition, we established
a LC–EC-MS platform advantageous in disulfide characterization
of complex and highly disulfide-bonded proteins such as human serum
albumin (HSA) by online EC reduction of nonreduced proteolytic digestions
Optimized Fast and Sensitive Acquisition Methods for Shotgun Proteomics on a Quadrupole Orbitrap Mass Spectrometer
Advances in proteomics are continually driven by the
introduction
of new mass spectrometric instrumentation with improved performances.
The recently introduced quadrupole Orbitrap (Q Exactive) tandem mass
spectrometer allows fast acquisition of high-resolution higher-energy
collisional dissociation (HCD) tandem mass spectra due to the parallel
mode of operation, where the generation, filling, and storage of fragment
ions can be performed while simultaneously measuring another ion packet
in the Orbitrap mass analyzer. In this study, data-dependent acquisition
methods for “fast” or “sensitive” scanning
were optimized and assessed by comparing stable isotope labeled yeast
proteome coverage. We discovered that speed was the most important
parameter for sample loads above 125 ng, where a 95 ms HCD scanning
method allowed for identification and quantification of more than
2000 yeast proteins from 1 h of analysis time. At sample loads below
125 ng, a 156 ms HCD acquisition method improved the sensitivity,
mass accuracy, and quality of data and enabled us to identify 30%
more proteins and peptides than the faster scanning method. A similar
effect was observed when the LC gradient was extended to 2 or 3 h
for the analysis of complex mammalian whole cell lysates. Using a
3 h LC gradient, the sensitive method enabled identification
of more than 4000 proteins from 1 μg of tryptic HeLa digest,
which was almost 200 more identifications compared to the faster scanning
method. Our results demonstrate that peptide identification on a quadrupole
Orbitrap is dependent on sample amounts, acquisition speed, and data
quality, which emphasizes the need for acquisition methods tailored
for different sample loads and analytical preferences
Comprehensive Identification of SUMO2/3 Targets and Their Dynamics during Mitosis
<div><p>During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.</p></div
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
Identification of mitotic SUMOylation targets using quantitative proteomics.
<p>A) Schematic outline of the protocol for growth and synchronization of cells in roller bottles. B) HeLa FRT TRex SUMO2 and HeLa FRT TRex SUMO2ΔGG cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by provoking mitotic exit by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against SUMO2/3, Cyclin B1 and Aurora A as markers for mitotic progression, and Vinculin. (<sup>★</sup>) indicates un-conjugated SUMO2 fusion proteins, (<sup>⧫</sup>) indicates endogenous SUMO2/3. C) Schematic outline of the quantitative proteomics strategy. Cells are grown in large-scale and synchronized in roller bottles as shown in A. SUMO2ΔGG cells were isotopically labelled with light “L”/R0K0 amino acids and arrested in prometaphase (grey), cells expressing SUMO2 were labelled with medium “M”/R6K4 amino acids and represented the mitotic exit stage one hour after ZM447439 addition (green), and cells expressing SUMO2 were labelled with heavy “H”/R10K8 amino acids and arrested in prometaphase (red). Equal amounts of cell lysate from the labelled populations were mixed and SUMO2 conjugated proteins were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100692#pone-0100692-g001" target="_blank">Figure 1</a>. The SUMO2 conjugate enriched sample was separated by SDS-PAGE, digested with trypsin and analyzed by mass spectrometry (MS). Lysates from the different experimental conditions were analyzed by western blotting antibodies against FLAG, Cyclin B1, Aurora A and Vinculin to confirm conjugation state and mitotic stage. (<sup>★</sup>) indicates un-conjugated SUMO2 fusion proteins. D) Scatter plot of the entire data set from screen I and screen II. The plot is showing the value of log<sub>2</sub>(M/L) and log<sub>2</sub>(H/L) SILAC ratios that are used to identify SUMOylation targets. The red dashed line at log<sub>2</sub>(M/L) = 1 and log<sub>2</sub>(H/L) = 1 represents the cut-off ratio of ≥2 for the respective SILAC pairs. Each point represents a single identified protein, proteins identified in screen I are illustrated in blue and proteins identified in screen II are in purple. Identified proteins that are classified as SUMOylation hits are above the dashed cut-off line and are darker colored, whereas identified proteins classified as background is below the cut-off and lighter. E) Diagram showing the number of identified targets (hits) of SUMO2/3 modification in screen I (blue), screen II (purple) and the overlap (dark blue) of hits that are identified in both. F) Scatter plot with the correlation between the screen I and screen II log<sub>2</sub>(H/M) ratios of identified SUMO2/3 target proteins. Each point represents a SUMO2/3 target. The pearson correlation, R, is shown.</p
RhoGDIα is specifically modified by SUMO in prometaphase.
<p>A) Identification of RhoGDIα as a SUMO2/3 target. SILAC ratios and MS data from screen I and II. B) Schematic representation of RhoGDIα with the amino acid sequence around lysine residues K138 and K141. The consensus modification motifs are indicated and the lysines are highlighted in red. C) Purification of Venus-RhoGDIα or Venus-RhoGDIα K138R/K141R from taxol arrested cells using GFP-trap beads. SUMO conjugation pathway components, Ubc9 and PIAS1-4, were depleted by RNAi, expression of Venus-RhoGDIα fusion proteins in the generated stable HeLa FRT Trex cell lines was induced with doxycycline and ZM447439 was added for one hour as indicated. The SUMOylation states of purified RhoGDIα and RhoGDIα K138R/K141R from the different experimental conditions were analyzed by western blotting with antibodies specific for SUMO2/3 and RhoGDIα. D) Parental HeLa FRT and HeLa FRT TRex SUMO2 cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by mitotic checkpoint override and progression by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against Cyclin B1, RhoGDIα, RhoA and α-tubulin, and shows that RhoGDIα itself is stable in mitosis. E) Using 3 different siRNA oligos for each target, normal HeLa cells were depleted for RhoGDIα or RhoGDIβ using RNAi. Lysates were analyzed for depletion efficiency and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. F) Stable HeLa FRT TRex FLAG-RhoGDIα or FLAG-RhoGDIα K138R/K141R cells were depleted for endogenous RhoGDIα and arrested in mitosis with taxol. Rescue with exogenous RhoGDIα fusion proteins were titrated in with increasing concentrations of doxycycline (ng/ml) as indicated. Lysates were analyzed for depletion efficiency, expression level of exogenous RhoGDIα and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. G) Representative still images from time-lapse movies of stable HeLa cell lines expressing Venus-RhoGDIα and Venus-RhoGDIα K138R/K141R as they progress through an unperturbed mitosis. The DIC and Venus channels are shown and the time of nuclear envelope breakdown (NEBD), metaphase and anaphase is indicated. H) As F) but using Venus-RhoGDIα.</p
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
Enrichment of SUMO2/3-conjugates through tandem-affinity purification.
<p>A) Sequence of the C-terminus of mature SUMO2 and the two mutants, SUMO2 Q87R and SUMO2ΔGG Q87R. The Q to R mutation is highlighted in red and the di-glycine conjugation motif in blue. B) Schematic representation of the generated SUMO2 Q87R and SUMO2ΔGG Q87R fusion constructs with the N-terminal 3*FLAG-His double affinity tag. C) Expression of SUMO2 fusion proteins were gradually induced with increased concentrations of doxycycline (ng/ml) added for 24 hours. SUMO2 fusion protein was conjugated to target proteins, whereas SUMO2ΔGG was not. (*) indicates un-conjugated SUMO2 fusion proteins. D) Schematic outline of the double-affinity purification procedure. Examples of the steps in Ni-NTA purification, buffer-change and anti-FLAG affinity purification are also shown on western blots with anti-FLAG primary antibody that detects SUMO2 fusion proteins. D1 = depletion 1. (*) indicates free 3*FLAG-His-SUMO2 fusion protein.</p
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
Complete Mapping of Complex Disulfide Patterns with Closely-Spaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry
Mapping
of disulfide bonds is an essential part of protein characterization
to ensure correct cysteine pairings. For this, mass spectrometry (MS)
is the most widely used technique due to fast and accurate characterization.
However, MS-based disulfide mapping is challenged when multiple disulfide
bonds are present in complicated patterns. This includes the presence
of disulfide bonds in nested patterns and closely spaced cysteines.
Unambiguous mapping of such disulfide bonds typically requires advanced
MS approaches. In this study, we exploited in-source reduction (ISR)
of disulfide bonds during the electrospray ionization process to facilitate
disulfide bond assignments. We successfully developed a LC-ISR-MS/MS
methodology to use as an online and fully automated partial reduction
procedure. Postcolumn partial reduction by ISR provided fast and easy
identification of peptides involved in disulfide bonding from nonreduced
proteolytic digests, due to the concurrent detection of disulfide-containing
peptide species and their composing free peptides. Most importantly,
intermediate partially reduced species containing only a single disulfide
bond were also generated, from which unambiguous assignment of individual
disulfide bonds could be done in species containing closely spaced
disulfide bonds. The strength of this methodology was demonstrated
by complete mapping of all four disulfide bonds in lysozyme and all
17 disulfide bonds in human serum albumin, including nested disulfide
bonds and motifs of adjacent cysteine residues