Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample

Shahinuzzaman, A. D. A.; Chakrabarty, Jayanta K.; Fang, Zixiang; Smith, David J.; Kamal, Abu Hena Mostafa; Chowdhury, Saiful M.

doi:10.1002/jssc.201901273

Cited by 14 publications

(16 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A high concentration of urea/thiourea/guanidine could reduce trypsin activity or introduce chemical modifications during proteolysis (e.g., carbamylation) [30]. Among the detergents, SDC was reported to improve protein denaturation and solubilization, enhancing the number of detected proteins [30][31][32][33][34]. The SDC detergent could be easily eliminated from the sample solution with acidic precipitation.…”

Section: Sample Preparation and Tryptic Digestion Optimizationmentioning

confidence: 99%

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

et al. 2021

View full text Add to dashboard Cite

The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092-5.222 μg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method's accuracy. The bias ranged from −2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 μg/L (bias −1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.

show abstract

Section: Sample Preparation and Tryptic Digestion Optimizationmentioning

confidence: 99%

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

et al. 2021

View full text Add to dashboard Cite

show abstract

“…In the XIC, the two precursors (M 2+ m/z 802.80 and M 3+ m/z 535.50) emerged in identical retention time (Supplemental Figure 3c and 3d) and identified to be the labeled lysozyme N-terminus by the tandem MS analysis (see Supplemental Table 1 for the detailed calculations). This experiment clearly shows methanol-chloroform purification is more advantageous over the commercial MWCO purification in removing the excess unreacted reagent to help enriching the protein, especially considering the widespread applications of methanol-chloroform in the purification of proteins from large-scale biological samples 37 .…”

Section: Evaluation Of Pfp-rink-biotin In the Enrichment And Identification Of Protein N-terminusmentioning

confidence: 91%

“…The α-amine labeling reaction was proceeded for 2 hours at 37 ˚C. The excess PFP-Rink-biotin was quenched by adding Tris-HCl buffer to a final concentration of 20 mM and removed by either 3K MWCO 48 or methanol-chloroform precipitation 37,49 , followed by the disulfide cleavages with DTT and alkylation with IAM in dark.…”

Section: Enrichment and Identification Of Standard Protein N-terminusmentioning

confidence: 99%

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

Fang¹,

Ms²,

Yepremyan³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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>

show abstract

“…The sample preparation for mass spectrometry in AP‐MS is fairly standard, involving the denaturation of proteins by using urea or guanidine hydrochloride, reduction of disulfide bonds, alkylation of free cysteine residues, and proteolytic digestion with trypsin or LysC [54, 62]. The preferred MS platform is implemented by LC‐MS/MS.…”

Section: Revealing Protein‐protein Interactions Using Affinity Purifimentioning

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample

Cited by 14 publications

References 29 publications

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

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

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

Contact Info

Product

Resources

About