Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry

Bhawal, Ruchika; Shahinuzzaman, A. D. A.; Chowdhury, Saiful M.

doi:10.1007/s13361-016-1503-0

Cited by 8 publications

(7 citation statements)

References 15 publications

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“…As demonstrated in the MS spectra of oxidized farnesyl KHSSGCAFL in Figure b, the mono-oxidized doubly charged peptide precursor ( m / z 585.43) generates an intense signature fragment ( m / z 458.27) after the neutral loss of RSOH. Compared to the MS 2 fragmentation pattern of the unoxidized prenyl peptides, the higher relative intensity of the signature fragment after RSOH loss demonstrates higher lability of mono-oxidized prenyl peptides in CID-MS 2 , which is consistent with the previous observation of RSOH neutral loss in MALDI-MS and ESI-MS systems. , MS 3 fragmentation of the RSOH neutral-loss-induced signature fragment again confirmed the same peptide sequence. Based on the MS-cleavable characteristics of both farnesyl and oxy-farnesyl peptides, a dual-stage NLMS3 (DS-NLMS3) strategy can be readily incorporated for targeting two different forms of the same prenylated peptide by specific neutral losses to enable their high-confidence identification (Figure c).…”

Section: Results and Discussionsupporting

confidence: 86%

“…More recently, an improved MS-cleavable strategy for identifying and differentiating prenylated peptides by oxidation and epoxidation was developed by our group . This method chemically converts the prenyl thioether linkage to the sulfoxide group, which turns out to be a more labile gas-phase cleavable group and consistent in generating a characteristic fragment after the neutral loss of RSOH (R = modified/unmodified prenyl side chain). , This simple oxidation reaction simultaneously alleviates the high hydrophobicity and opens new opportunity to incorporate enrichment tags on the prenylated peptides for targeted enrichment.…”

Section: Introductionmentioning

confidence: 99%

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Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

Fang

Chowdhury

2021

Anal. Chem.

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Protein prenylation is an important post-translational modification that regulates protein interactions, localizations, and signaling pathways in normal functioning of eukaryotic cells. It is also a critical step in the oncogenic developments of various cancers. Direct identification of native protein prenylation by mass spectrometry (MS) has been challenging due to high hydrophobicity and the lack of an efficient enrichment technique. Prior MS studies of prenylation revealed that prenyl peptides readily generate high-intensity fragments after neutral loss of the prenyl group (R group), and more recent investigation of oxidized prenyl peptides discovered more consistent neutral loss of the oxidized prenyl group (RSOH group). Here, a dual-stage neutral loss MS 3 (DS-NLMS3)-based strategy is therefore developed by combining both gas-phase cleavable properties of the prenyl thioether bond and mono-oxidized thioether to improve the large-scale identification of prenylation. Both neutral losses can individually and distinctively confirm the prenylation type in MS 2 and the sequence of the prenyl peptide upon targeted MS 3 fragmentation. This dual-faceted NLMS3 strategy significantly improves the confidence in the identification of protein prenylation from large-scale samples, which enables the unambiguous identification of prenylated sites of the spiked low-abundance farnesyl peptide and native prenyl proteins from mouse macrophage cells, even without prior enrichment during sample preparation. The ease of incorporating this strategy into the prenylation study workflow and minimum disruption to the biological lipidome are advantageous for unraveling unknown native protein prenylation and further developments in profiling and quantifying prenylome.

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Section: Results and Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

Fang

Chowdhury

2021

Anal. Chem.

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“…In terms of directly targeting protein prenylation, our group had reported a strategy to alleviate the high hydrophobicity issue by oxidizing the prenyl thioether linkage and epoxidizing the unsaturated prenyl groups, which can simultaneously generate a signature prenyl‐dependent neutral loss in tandem mass spectrum for differentiating farnesylation and geranylgeranylation more confidently [45, 46]. The epoxy group also helps to increase the chemical reactivity of prenyl groups, which could potentially be more easily targeted for the direct derivatization and selective enrichment of protein prenylation (Figure 1).…”

Section: Lipid Post‐translational Modificationsmentioning

confidence: 99%

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

Zacharias

Fang

Rahman

et al. 2020

J of Separation Science

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Protein post‐translational modifications and protein interactions are the central research areas in mass‐spectrometry‐based proteomics. Protein post‐translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome‐wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross‐linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post‐translational modification or protein interaction network.

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“…MS-cleavable reagents are increasingly gaining popularities in the research of low-abundant proteins and protein PTMs by producing unique signatures such as signature ion or neutral losses upon tandem MS fragmentation, which assist in the unambiguous identifications 26,28 and more precise quantifications 29 of these otherwise overshadowed targets. This unique feature of MScleavable reagent is an attractive strategy for identifying the protein N-terminus which are dramatically outnumbered by internal peptides.…”

Section: Development Of Ms-cleavable Reagent Pfp-rink-biotin For N-terminus Labelingmentioning

confidence: 99%

A Mass Spectrometry-Cleavable N-Terminal Tagging Strategy for System-Level Protease Activity Profiling

Fang¹,

Ms²,

Yepremyan³

et al. 2020

Preprint

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Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. This irreversible PTM is regulated and catalyzed by proteases through hydrolysis reaction in the process of protein maturation or degradation. The identification of proteolytic substrates is pivotal in understanding the specificity of proteases and the physiological role of proteolytic process. However, tracking these biological phenomena in native cells is very difficult due to their low abundances. Currently, no efficient methods are available to identify proteolytic products from large-scale samples. To facilitate the targeted identification of low-abundant proteolytic products, we devise a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-terminus. Within the PFP-Rink-biotin reagent, an MS-cleavable feature is designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. This is the first reported study of identifying proteolytic N-terminus by MS-cleavable feature widely adopted in studying low-abundant protein PTMs and cross-linking/MS. The proof-of-concept study was performed with multiple standard proteins whose N-terminus were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia.coli cells were demonstrated along with a bioinformatics workflow. We believe this method can be very useful to locate proteolytic products in native cellular environment with high confidence.<br>

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Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry

Cited by 8 publications

References 15 publications

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

A Mass Spectrometry-Cleavable N-Terminal Tagging Strategy for System-Level Protease Activity Profiling

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