In‐gel digestion with endoproteinase LysC

Wada, Yoshinao; Kadoya, Machiko

doi:10.1002/jms.384

Cited by 10 publications

(7 citation statements)

References 3 publications

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“…The eluate containing S ‐carbamidomethylated haptoglobin (1 mL, in 0.05 N HCl) was immediately neutralized with 100 μL of 1 M Tris‐HCl (pH 9.0). The neutralized solution was mixed with 20 μL of 50 mM Tris‐HCl (pH 8.5) containing an enzyme mixture of lysylendopeptidase (2 μg) and trypsin (2 μg) and incubated for 16 hr at 37°C 20. After boiling, the solution was evaporated to dryness.…”

Section: Methodsmentioning

confidence: 99%

Site‐specific analysis of N‐glycans on haptoglobin in sera of patients with pancreatic cancer: A novel approach for the development of tumor markers

Nakano¹,

Nakagawa²,

Ito³

et al. 2008

Intl Journal of Cancer

133

157

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It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N-glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N-glycans. In the present study, site-specific N-glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di-, tri-and tetra-branched glycans of haptoglobin increased in the sera of PC patients. Tri-antennary N-glycans containing a Lewis X-type fucose markedly increased at the Asn211 site of haptoglobin Nglycans. While fucosylated N-glycans derived from serum haptoglobin of patients with CP slightly increased, di-fucosylated tetraantennary N-glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site-specific analyses of N-glycans may be useful as novel tumor markers for PC.

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Section: Methodsmentioning

confidence: 99%

Site‐specific analysis of N‐glycans on haptoglobin in sera of patients with pancreatic cancer: A novel approach for the development of tumor markers

Nakano¹,

Nakagawa²,

Ito³

et al. 2008

Intl Journal of Cancer

133

157

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“…In the case of Rho130, the high levels of Arg residues precluded the use of trypsin due to extensive fragmentation. Consequently, we utilized LysC, a protease that cleaves C-terminal to Lys residues, to generate peptides . For unmodified Rho130, three distinct peptides were detected, Asn8-Lys31 (cleavage at Lys7 and Lys31), Gln32-Lys40 (cleavage at Lys31 and Lys40), and Ser45-Lys100 (cleavage at Lys44 and Lys100), providing coverage over five of the Lys residues (Lys7, Lys31, Lys40, Lys44, and Lys100).…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, we utilized LysC, a protease that cleaves C-terminal to Lys residues, to generate peptides. 50 For unmodified Rho130, three distinct peptides were detected, Asn8-Lys31 (cleavage at Lys7 and Lys31), Gln32-Lys40 (cleavage at Lys31 and Lys40), and Ser45-Lys100 (cleavage at Lys44 and Lys100), providing coverage over five of the Lys residues (Lys7, Lys31, Lys40, Lys44, and Lys100). The ESI MS of Rho130−initiator conjugate LysC peptides indicated essentially 100% cleavage at Lys7 and partial cleavage at Lys31 (not shown).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Protein Dynamics Is Altered by a High Surface Density of Atomic Transfer Radical Polymerization Polymers

2021

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The effect of atomic transfer radical polymerization (ATRP) polymers on the structure and dynamics of a 14.5 kDa RNA binding protein, Rho130, was assessed using NMR. A nearhomogeneous sample was generated by optimizing initiator coupling to maximize the number of modified Lys residues. The reactivity of individual Lys residues was correlated with the average solvent accessible surface area from molecular dynamics (MD) simulations and influenced by local interactions. Larger structural changes were seen with the addition of the initiator alone than with polymer growth. Structural changes were localized to the Nterminal helical domain of the protein and MD simulations suggest stabilization of the terminus of one helix by the addition of the ATRP initiator and an initiator-induced change in interhelical angles. Relaxation dispersion shows that polymer addition, but not attachment of the initiator, causes a reduction in the microsecond−millisecond dynamics of the hydrophobic core.

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“…There are additionally the so-called 'sequencing endoproteases' [14], which include Lys-C, which cleaves only C-terminal to lysine residues, Arg-C, Glu-C and Asp-N. Used in conjunction with trypsin, these proteases also yield improved sequence coverage [4]. Disadvantages of using these endoproteases include their relatively high cost, catalytic inefficiency and that digestion with the alternative proteases can result in peptides that lack the Cterminal basic residue typically responsible for the intense y-ion series.…”

Section: Figure 1 the Increase In Proteomics Publicationsmentioning

confidence: 99%

The proteomic future: where mass spectrometry should be taking us

Thelen

Miernyk

2012

Biochemical Journal

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A newcomer to the -omics era, proteomics, is a broad instrument-intensive research area that has advanced rapidly since its inception less than 20 years ago. Although the 'wet-bench' aspects of proteomics have undergone a renaissance with the improvement in protein and peptide separation techniques, including various improvements in two-dimensional gel electrophoresis and gel-free or off-gel protein focusing, it has been the seminal advances in MS that have led to the ascension of this field. Recent improvements in sensitivity, mass accuracy and fragmentation have led to achievements previously only dreamed of, including whole-proteome identification, and quantification and extensive mapping of specific PTMs (post-translational modifications). With such capabilities at present, one might conclude that proteomics has already reached its zenith; however, 'capability' indicates that the envisioned goals have not yet been achieved. In the present review we focus on what we perceive as the areas requiring more attention to achieve the improvements in workflow and instrumentation that will bridge the gap between capability and achievement for at least most proteomes and PTMs. Additionally, it is essential that we extend our ability to understand protein structures, interactions and localizations. Towards these ends, we briefly focus on selected methods and research areas where we anticipate the next wave of proteomic advances.

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In‐gel digestion with endoproteinase LysC

Cited by 10 publications

References 3 publications

Site‐specific analysis of N‐glycans on haptoglobin in sera of patients with pancreatic cancer: A novel approach for the development of tumor markers

Site‐specific analysis of N‐glycans on haptoglobin in sera of patients with pancreatic cancer: A novel approach for the development of tumor markers

Protein Dynamics Is Altered by a High Surface Density of Atomic Transfer Radical Polymerization Polymers

The proteomic future: where mass spectrometry should be taking us

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