Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Chang, Chorng‐Ping; Liao, Hsin-Kai; Juo, Chiun-Gung; Chen, Shuhua; Chen, Yu‐Ju

doi:10.1016/j.aca.2005.07.025

Cited by 12 publications

(11 citation statements)

References 41 publications

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“…However, as for in-gel digestion, the recovery of large peptides was very low. An alternative approach directly analyzed the proteins or the peptides adsorbed onto the membrane by MALDI-MS [87]. In this approach, it is crucial that the matrix solution can dissolve the nitrocellulose-bound proteins or peptides in order for them to be incorporated into the matrix crystals.…”

Section: On-membrane Digestionmentioning

confidence: 99%

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

2015

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Despite enormous advances in the mass spectrometry and proteomics fields during the last two decades, the analysis of membrane proteins still remains a challenge for the proteomic community. Membrane proteins play a wide number of key roles in several cellular events, making them relevant target molecules to study in a significant variety of investigations (e.g., cellular signaling, immune surveillance, drug targets, vaccine candidates, etc.). Here, we critically review the several attempts that have been carried out on the different steps of the sample preparation procedure to improve and modify existing conventional proteomic strategies in order to make them suitable for the study of membrane proteins. We also revise novel techniques that have been designed to tackle the difficult but relevant task of identifying and characterizing membrane proteins.

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Section: On-membrane Digestionmentioning

confidence: 99%

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

2015

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“…In a previous study, we demonstrated that PVDF-aided MALDI-TOF MS can be an effective approach for improvement of the spot-to-spot variation in MALDI detection and profiling and characterizing membrane proteins [31]. Compared with the standard metallic MALDI sample plate, both intensity and S/N ratio were greatly improved in MALDI mass spectra by using PVDF membrane.…”

Section: Introductionmentioning

confidence: 99%

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI‐TOF MS

Chen¹,

Liao²,

Chang³

et al. 2007

Proteomics

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Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.

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“…Chang et al [29] separated membrane proteins from gram-negative bacteria by SDS ⁄ PAGE, electroblotted the proteins onto the Hybond-P PVDF membranes and then fixed the membrane to the sample target using conductive double-sided adhesive tape. The matrix for MALDI-TOF MS was added to the samples for MS analysis.…”

Section: Membrane Namementioning

confidence: 99%

“…Therefore, it was necessary to develop new membranes on which digests of the immobilized protein can be ionized efficiently. Without this improvement, peptides obtained by on-membrane digestion of gel-resolved proteins would have to be eluted from the membrane and then subjected to MALDI for identification [25][26][27][28][29][30].…”

Section: Small Pore Size Of Membrane Increases Ion Yield In Msmentioning

confidence: 99%

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

Ino

Hirano

2011

The FEBS Journal

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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.

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Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Cited by 12 publications

References 41 publications

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI‐TOF MS

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

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