Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome

Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome

Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome

Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome

Temporal Profiling Establishes a Dynamic <i>S</i>‑Palmitoylation Cycle

<i>S</i>-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling <i>S</i>-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic <i>S</i>-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal <i>S</i>-palmitoylation assay for temporal analysis of <i>S</i>-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies <i>S</i>-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state <i>S</i>-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. <i>S</i>-palmitoylation profiling of APT1^–/–/APT2^–/– mouse brains similarly show no change in <i>S</i>-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic <i>S</i>-palmitoylated proteins, raising the possibility that <i>S</i>-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state <i>S</i>-palmitoylation across the proteome

DIA-SIFT: A Precursor and Product Ion Filter for Accurate Stable Isotope Data-Independent Acquisition Proteomics

Quantitative mass spectrometry-based protein profiling is widely used to measure protein levels across different treatments or disease states, yet current mass spectrometry acquisition methods present distinct limitations. While data-independent acquisition (DIA) bypasses the stochastic nature of data-dependent acquisition (DDA), fragment spectra derived from DIA are often complex and challenging to deconvolve. In-line ion mobility separation (IMS) adds an additional dimension to increase peak capacity for more efficient product ion assignment. As a similar strategy to sequential window acquisition methods (SWATH), IMS-enabled DIA methods rival DDA methods for protein annotation. Here we evaluate IMS-DIA quantitative accuracy using stable isotope labeling by amino acids in cell culture (SILAC). Since SILAC analysis doubles the sample complexity, we find that IMS-DIA analysis is not sufficiently accurate for sensitive quantitation. However, SILAC precursor pairs share common retention and drift times, and both species cofragment to yield multiple quantifiable isotopic <i>y</i>-ion peak pairs. Since <i>y</i>-ion SILAC ratios are intrinsic for each quantified precursor, combined MS1 and <i>y</i>-ion ratio analysis significantly increases the total number of measurements. With increased sampling, we present DIA-SIFT (<i>S</i>ILAC <i>I</i>ntrinsic <i>F</i>iltering <i>T</i>ool), a simple statistical algorithm to identify and eliminate poorly quantified MS1 and/or MS2 events. DIA-SIFT combines both MS1 and <i>y</i>-ion ratios, removes outliers, and provides more accurate and precise quantitation (<15% CV) without removing any proteins from the final analysis. Overall, pooled MS1 and MS2 quantitation increases sampling in IMS-DIA SILAC analyses for accurate and precise quantitation

Targeted Annotation of S‑Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry

Protein S-sulfinylation (R–SO₂^–) and S-sulfonylation (R–SO₃^–) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 μm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site

HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme–substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, <i>p</i>-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical crosslinking approach identified novel HDAC8-specific substrates with high catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery

HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme–substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, <i>p</i>-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical crosslinking approach identified novel HDAC8-specific substrates with high catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs