Direct on-membrane peptide mass fingerprinting with MALDI–MS of tyrosine-phosphorylated proteins detected by immunostaining

Nakanishi, Takashi; Ando, Eiji; Furuta, Masaru; Tsunasawa, Susumu; Nishimura, Osamu

doi:10.1016/j.jchromb.2006.08.024

Cited by 9 publications

(10 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It can be envisaged that MS/MS generally results in more reliable protein identifications than single MS approaches. In addition to ingel digestion, tryptic digests can be performed on the pieces of membranes that contain the blotted antigenic protein of interest [20].…”

Section: Maldi-tof Ms(/ms)mentioning

confidence: 99%

Immunoproteomics: From biomarker discovery to diagnostic applications

Tjalsma

Schaeps

Swinkels

2008

Proteomics Clinical Apps

View full text Add to dashboard Cite

Circulating antibodies reflect a molecular imprint of antigens that are related to autoimmune diseases, cancer or infection. Importantly, serum antibodies are useful clinical markers as they carry diagnostic information from all around the human body. Moreover, the amplification cascade governed by the humoral immune system causes a surplus of circulating antibodies after appearance of the corresponding (low abundance) antigen. In combination with the fact that antibodies are highly stable compared to many other serum proteins, they seem ideal to be implemented in clinical diagnostic assays for the detection of antigen-associated diseases. This review summarises advances in immunoproteomics with respect to technologies for biomarker discovery, with special emphasis on recently developed gel-free MS-based approaches, and looks forward to potential immunoproteomic applications in diagnostic medicine.

show abstract

Section: Maldi-tof Ms(/ms)mentioning

confidence: 99%

Immunoproteomics: From biomarker discovery to diagnostic applications

Tjalsma

Schaeps

Swinkels

2008

Proteomics Clinical Apps

View full text Add to dashboard Cite

show abstract

“…After SDS‐PAGE, the bands of the degenerated C‐PC were cut off and destained with 50% ACN/25 mM NH 4 HCO 3 , and then reduced with 10 mM DTT at 56°C and alkylated in the dark with 50 mM iodoacetamide at room temperature for 1 h. After that, the gel plugs were lyophilized and immersed in 15 mL of 10 ng/mL trypsin solution in 25 mM NH 4 HCO 3 , and the digestion was kept at 37°C for 15 h. Tryptic peptide mixtures were first extracted with 100 mL 5% TFA and then with same volume of 2.5% TFA/50% ACN. The extracted solutions were blended, lyophilized, and used for MS analysis 23 using the following conditions: Positively charged ions were analyzed in reflection mode, using delayed extraction. Typically, 100 shots were averaged to improve the signal‐to‐noise ratio.…”

Section: Methodsmentioning

confidence: 99%

Orthogonal test design for optimization of suitable conditions to separate C‐phycocyanin from Spirulina platensis by high‐speed counter‐current chromatography using reverse micelle solvent system

Yin

et al. 2011

J of Separation Science

View full text Add to dashboard Cite

High-speed counter-current chromatography (HSCCC) was applied to separate C-phycocyanin (C-PC) from Spirulina platensis in the article. The suitable conditions were optimized by an orthogonal test design (L(9)(3)(3)), including the stationary phase of reverse micelle solvent system (0.10 g/mL cetyltrimethylammonium bromide [CTAB]/isooctane-hexylalcohol), mobile phase A (0.05 mol/L sodium phosphate buffer, pH 4.0, containing 0.2 mol/L KCl) and mobile phase B (0.05 mol/L sodium phosphate buffer, pH 8.0, containing 0.4 mol/L KCl). Under the selected conditions, 78.7 mg protein was purified from 200 mg crude extract of S. platensis, and the purity of the product was 4.25 based on the absorbance ratio of A(620)/A(280) , which was increased 6.85 times compared with the crude extract. Then, the protein was identified to be C-PC by MALDI-TOF/TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis compared with the standard. The application of HSCCC used in the separation of C-PC from S. platensis was first reported in the article. Furthermore, three kinds of tumor cell lines including human hepatoma cell line SMMC-7721, human ovarian carcinoma cell line ES-2, and human lung adenocarcinoma cell line SPCA-1 were used to evaluate the anticancer activities of the separated product, and the results showed that the separated C-PC had excellent anti-tumor actions with the IC(50) values at 2.998, 4.854, and 8.423 μg/mL, respectively, for 48 h treatment. The outcome indicates that an effective method for C-PC purification by HSCCC has been established.

show abstract

“…[33] and Nakanishi et al. [34,35] used the same piezoelectric chemical inkjet printer for the enzymatic digestion of proteins on membranes. Kimura et al.…”

Section: Proteins Blotted Onto the Membrane Filters Can Be Analyzed Bmentioning

confidence: 99%

“…The gel-resolved proteins can be characterized by MALDI-MS. Sloane et al [32] immobilized gelresolved proteins onto Immobilon P SQ PVDF and nitrocellulose membranes, and digested the proteins with proteases on membranes using a piezoelectric chemical inkjet printer, and then they identified antigens and glycoproteins using MS. Kimura et al [33] and Nakanishi et al [34,35] used the same piezoelectric chemical inkjet printer for the enzymatic digestion of proteins on membranes. Kimura et al [33] electroblotted gel-resolved proteins onto Immobilon P SQ Table 1.…”

Section: On-membrane Characterization Of Gel-resolved Proteins By Msmentioning

confidence: 99%

Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

Ino

Hirano

2011

The FEBS Journal

View full text Add to dashboard Cite

In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed. Proteomic analyses begin with protein expression profilingProteome research involves the comprehensive analysis of protein expression profiles, the function and functional networks of proteins, and the relationship between proteins and diseases. Upgraded protein databases, and highly sensitive, accurate and high-throughput analytical techniques, such as mass spectrometry (MS), have greatly facilitated and accelerated the development of proteome research.Two standard methods, the protein shotgun method [1] and the 2D electrophoresis (2DE)-MS ⁄ MS method [2], are commonly used in the first step of proteome analysis. In the shotgun method, a sample containing a number of proteins is digested with proteases such as trypsin, which has comparatively high substrate specificity. These digested proteins are separated by liquid chromatography (LC), analyzed by MS ⁄ MS and then identified based on their sequence information. In contrast, in the 2DE-MS ⁄ MS method, most proteins are separated by 2DE, protease-digested in gels and then analyzed by MS [3]. Next, the proteins are identified based on peptide mass fingerprints or on sequence information obtained by MS ⁄ MS and database searches [4].Both methods can profile the expression of a number of proteins. However, the shotgun method has been more frequently used for recent proteomic analyses. The shotgun method typically identifies more proteins than 2DE-MS ⁄ MS. In addition, the shotgun method can be easily automated and therefore does not require professional skills for the analysis. In contrast, 2DE is laborious, and it is not easy for beginners to obtain reproducible results, particularly with the 'in-gel' digestion of proteins after electrophoresis. However, the shotgun method cannot completely replace 2DE-MS ⁄ MS for proteomic studies because 2DE-MS ⁄ MS has several advantages over the shotgun method, as described below.

show abstract

Direct on-membrane peptide mass fingerprinting with MALDI–MS of tyrosine-phosphorylated proteins detected by immunostaining

Cited by 9 publications

References 21 publications

Immunoproteomics: From biomarker discovery to diagnostic applications

Immunoproteomics: From biomarker discovery to diagnostic applications

Orthogonal test design for optimization of suitable conditions to separate C‐phycocyanin from Spirulina platensis by high‐speed counter‐current chromatography using reverse micelle solvent system

Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

Contact Info

Product

Resources

About