Collision Energy Optimization of b- and y-Ions for Multiple Reaction Monitoring Mass Spectrometry

Multiple reaction monitoring (MRM) is a highly sensitive and increasingly popular method of targeted mass spectrometry (MS) that can be used to selectively detect and quantify peptides and their corresponding proteins of interest within biological samples. The sensitivity of MRM-MS is highly dependent upon the tuning of transition-specific parameters, especially the collision energy (CE) applied during peptide fragmentation. Currently, empirical equations for CE work best for y-type ions and are much less effective for other types of transitions, such as b-type ions and small y-type transitions across particular amide bonds, which could also be useful for MRM-MS if optimized for maximum signal transmission. In this work, we have performed a CE optimization of all transitions for 80 doubly charged peptides, the results of which were used to define separate CE equations for b-ions and y-ions, as well as for small y-type ions derived from the fragmentation of amide bonds bounded on the amino-terminal side by aspartic or glutamic acid residues (D/E-X transitions). This analysis yielded four major observations: (1) b-ions tend to require lower collision energies than y-ions for optimal fragmentation, while D/E-X transitions tend to require more; (2) CE equations predict the optimal CEs more closely when product ion m/z dependence is included, in addition to the current standard of precursor ion m/z dependence; (3) separate CE equations for y-ions, b-ions, and D/E-X transitions are more effective than the previous one-size-fits-all equations, but best results are achieved by optimizing transitions individually; and (4) while b-ions gain substantial signal from CE optimizationoften increases of several-foldthey still tend to rank lower than y-ions from the same peptide. These results confirm the notion that y-ions are usually the first-choice transitions for MRM experiments but also demonstrate, for the first time, that b-ions can be viable targets as well, if the proper collision energies are used

Kinetic Analysis of BCL11B Multisite Phosphorylation–Dephosphorylation and Coupled Sumoylation in Primary Thymocytes by Multiple Reaction Monitoring Mass Spectroscopy

Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation–dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five residues apart

Kinetic Analysis of BCL11B Multisite Phosphorylation–Dephosphorylation and Coupled Sumoylation in Primary Thymocytes by Multiple Reaction Monitoring Mass Spectroscopy

Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation–dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five residues apart

¹³C- and ¹⁵N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in <i>C</i>. <i>elegans</i>

<div><p>Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in <i>C</i>. <i>elegans</i> combining ¹³C and ¹⁵N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with <i>SCD</i> RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.</p></div

Characterization of SCDs as Membrane Maintenance Regulators.

<p>(A) The major FAs in <i>C</i>. <i>elegans</i> are produced by the elongation and desaturation pathway shown here. An alternate pathway downstream of FA synthase <i>(fasn-1)</i> is used for the production of monomethyl-branched chain FAs, C15iso and C17iso. (B) The amount of new FA incorporated each hour was quantified in animals treated with adult-only RNAi against the FA synthesis, elongation and desaturation genes for 48 hours. The resulting data are summarized here with species with less replenishment than control RNAi colored light blue (>25% decrease) and dark blue (>50% decrease). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s001" target="_blank">S1 Dataset</a> for complete source data. (C) To determine the overall impact on membrane replenishment, we averaged the replacement rates for each FA species and normalized by the abundance of each species in the PL population. RNAi of <i>pod-2</i>, <i>fat-5</i>, <i>fat-6</i> and <i>fat-7</i> resulted in significant reduction in overall membrane maintenance. (D) The relative amount of synthesized FA is reduced with <i>fat-7</i> RNAi treatment (blue) compared to control RNAi (black). Numbers shown represent the mean of at least three experiments ± SEM. Statistical significance was determined by two-tailed unpaired t-tests (*p<0.05, **p<0.01, ***p<0.001).</p

Quantification of Membrane Dynamics With ¹³C-Labeling and GC-MS in Young Adults.

<p>(A) Day 3 adult nematodes were fed a diet of 50% ¹³C-labeled <i>E</i>. <i>coli</i> for 6 hours to introduce ¹³C into their lipids and mark them as newly added or modified. The ¹³C can be traced into the major membrane FAs (>2% abundance) by GC-MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s011" target="_blank">S1 Table</a>). The amount of new FA measured by ¹³C-incorporation, was determined for purified PLs (black) and NLs (white). C20s with 4 or more double bonds were excluded in the analysis due to insufficient isotopomers. C20:0 and C20:2 FAs were profiled despite low abundance in the membrane to increase the representation of long-chain FAs. (B) After combining the amount of new FA found in all major C15 to C19 FAs, the overall contribution of new FA/hour was significantly greater in the PLs versus the NLs. (C) The relative amount of new FA derived from <i>de novo</i> FA synthesis was not significantly different for PLs (black) and NLs (white) except for C16:0. Numbers shown represent the mean of at least four experiments ± SEM. Statistical significance was calculated by two-tailed unpaired t-tests where significance (*p<0.05, **p<0.01, ***p<0.001) is noted by the asterisks.</p

Dietary ¹³C Allows for Modeling of Individual FA Dynamics.

<p>(A) Day 3 adult nematodes are grown on ¹²C (gray)-<i>E</i>.<i>coli</i> (OP50) before the diet is switched to 50% ¹³C (green)-<i>E</i>. <i>coli</i>. After 6 hours, the C18:1n7 in the nematode was a combination of old fat from the original (pre-¹³C) diet, FA absorbed directly from the diet, and FA derived from lipogenesis with a random but statistically definable incorporation of single carbon molecules. (B) The C18:1n7 isotopomers were assessed by GC-MS. The natural abundance of ¹³C in the environment as well as background were subtracted from the C18:1n7 isotopomers derived from ¹³C-fed animals. The contributions of <i>de novo</i> FA synthesis (light green) and dietary absorption (dark green) can be mathematically determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#sec014" target="_blank">Material and Methods</a>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.ref009" target="_blank">9</a>]). (C) For any given fatty acid species including the C18:1n7 shown here, the total abundance of the fatty acid is determined by integrating the area under its peak in the gas chromatograph (total abundance outlined in dashed line). Further, we can divide the area as follows with gray representing ¹²C species and green indicating the presence of at least one ¹³C molecule. The unlabeled fatty acids absorbed during the labeling period were accounted for as new. The percentage of the population from absorption (dark green) and synthesis (light green) can be quantified to ultimately define that 29.3 ± 1.5% of the C18:1n7 peak is generated from new fatty acid.</p

LC-MS/MS Analysis of PL Composition in <i>fat-7</i> RNAi Treated Animals.

<p>The major PL species (>2%) in control animals are represented by X:Y (where X is the number of carbons in both FAs tails and Y indicates the total double bonds). For a complete list of species, see source data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s002" target="_blank">S2 Dataset</a>). For P-PlsEtn, the LC-MS/MS detection allowed for both chains to be accurately reported. Control RNAi is represented with white bars, and <i>fat-7</i> RNAi is shown in red. The fatty acid tails for each species can be qualitatively assessed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s009" target="_blank">S7 Fig</a>). Significant changes as are indicated by * (p<0.05) and ** (p<0.01) determined by two-tailed unpaired t-tests. Numbers shown represent the mean of at least three experiments ± SEM.</p

Modeling the Impact of <i>SCD</i> Depletion on Membrane Dynamics.

<p>(A) ¹³C-tracers allow for the quantification of <i>de novo</i> synthesis as well as incorporation of FAs into PLs (measurements from ¹³C-tracers shown in green). Our implementation of ¹⁵N to measure PL headgroup turnover defines the rates that the two most abundant species within each major PL class are replaced (shown in blue); for example, 40:10 and 38:6 are the largest contributors to the PtdCho pool and are replaced at a rate of 1.3% new PL/hour and 1.5% new PL/hour respectively. A weighted average of the PL replacement calculated for the PL species reported here predicts that 1.5% of the total PLs are replaced each hour. We combined the PL replacement rates with those calculated for FA tails where we found that 4.5% of measured fatty acid tails are new each hour. The quantification after 24 hours revealed a stable population of membrane lipid (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s005" target="_blank">S3 Fig</a>); thus, we reduced our average fatty acid predictions by 30% to account for that population. Taken together, we use the ¹³C and ¹⁵N-tracers to define how the individual lipid components (FA tails and polar headgroups) are replaced over time, and predict that at least 60% of the membrane is new each day when considering each fatty acid tail and headgroup as individual entities. (B) Upon <i>fat-7</i> RNAi treatment, there is a buildup of C18:0 and a depletion of C18:1n9, which in turn reduces the activation of <i>de novo</i> FA synthesis. Subsequently, FAs provided to PL synthesis and ultimately provided to the membrane, are severely compromised.</p