BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow

Takemori, Ayako; Ishizaki, Jun; Nakashima, Kenji; Shibata, Takeshi; Kato, Hidemasa; Kodera, Yasuhiro; Suzuki, Tetsuro; Hasegawa, Hitoshi; Takemori, Nobuaki

doi:10.1021/acs.jproteome.0c00749

Cited by 17 publications

(12 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While the original concept of a ‘molecular scanner’ [ 139 ] might in theory address this concern it would, minimally, still require genuine quantitative recovery (i.e., transfer) of all proteoforms resolved in the 2D gel. Furthermore, while dissolvable polyacrylamide matrices have been known for several decades [ 140 , 141 ], the chemicals needed would likely result in nonspecific and non-native alterations to the proteome.…”

Section: Discovery Proteomicsmentioning

confidence: 99%

Proteomes Are of Proteoforms: Embracing the Complexity

2021

View full text Add to dashboard Cite

Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must assess the myriad of proteoforms that arise at any point before, after, and between translation and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as newly defined species. There are numerous analytical methods currently used to address proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. We must embrace the complexity. Yes, these are the hard questions, and this will not be easy…but where is the fun in easy?

show abstract

Section: Discovery Proteomicsmentioning

confidence: 99%

Proteomes Are of Proteoforms: Embracing the Complexity

2021

View full text Add to dashboard Cite

show abstract

“…A widely used MW-based separation technique for intact proteins preferred for its ease is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A separation strategy combining SDS-PAGE and reversed-phase LC, called GeLC–MS, was originally developed for bottom-up proteomics (BUP) to analyze peptides from in-gel digested proteins. − In contrast, the application of GeLC–MS to TDP has long been hampered by the low recovery of proteins as intact species from polyacrylamide gels, but significant progress has recently been made with the development of PEPPI-MS ( P assively E luting P roteins from P olyacrylamide gels as I ntact species for MS ), an efficient passive extraction method for intact proteins in gels …”

Section: Introductionmentioning

confidence: 99%

Size-Based Proteome Fractionation through Polyacrylamide Gel Electrophoresis Combined with LC–FAIMS–MS for In-Depth Top-Down Proteomics

et al. 2022

Self Cite

View full text Add to dashboard Cite

The combination of liquid chromatography (LC) and gas-phase separation by field-asymmetric ion mobility spectrometry (FAIMS) is a powerful proteoform separation system for top-down proteomics. Here, we present an in-depth top-down proteomics workflow, GeLC–FAIMS–MS, in which a molecular-weight-based proteome fractionation approach using SDS-polyacrylamide gel electrophoresis is performed prior to LC–FAIMS–MS. Since individual bands and their corresponding mass ranges require different compensating voltages (CVs), the MS parameters for each gel band and CV were optimized to increase the number and reliability of proteoform identifications further. We developed an easy-to-implement and inexpensive procedure combining the earlier established Passively Eluting Proteins from Polyacrylamide gels as Intact species (PEPPI) protocol with an optimized Anion-Exchange disk-assisted Sequential sample Preparation (AnExSP) method for the removal of stains and SDS. The protocol was compared with a methanol–chloroform–water (MCW)-based protein precipitation protocol. The results show that the PEPPI-AnExSP procedure is better suited for the identification of low-molecular-weight proteoforms, whereas the MCW-based protocol showed advantages for higher-molecular-weight proteoforms. Moreover, complementary results were observed with the two methods in terms of hydrophobicity and isoelectric points of the identified proteoforms. In total, 8500 proteoforms could be identified in a human proteome standard, showing the effectiveness of the gel-based sample fractionation approaches in combination with LC–FAIMS–MS.

show abstract

“…1 A simple, fast, low-cost, and reliable diagnostic clinical test for CEA is much needed for detection and regular monitoring of the prognosis of colorectal cancer. In this regard, many attempts have been made for the detection of CEA by various methods including enzyme-linked immunosorbent assays, 2 colorimetry, 3 mass spectroscopy, 4 gel electrophoresis, 5 surface plasmon resonance, 6 and electrochemiluminescence. 7 Combination of the robustness of immunoassay and the simplicity with the inherent high sensitivity of the electrochemical method exhibits a potential strategy for reliable biosensor development.…”

Section: ■ Introductionmentioning

confidence: 99%

Morphologically Flex Sm-MOF Based Electrochemical Immunosensor for Ultrasensitive Detection of a Colon Cancer Biomarker

Biswas

Lan

et al. 2022

Anal. Chem.

View full text Add to dashboard Cite

Despite having the potential to synthesize stable metal−organic frameworks (MOFs), rare earth metal-based MOFs have not been exploited extensively. Owing to the high coordination numbers, the MOFs can generate a suitable coordination environment for various applications. Herein, samarium (Sm)-based MOFs were synthesized with three different organic linkers, namely, trimesic acid (TMA), meso-tetra(4carboxyphenyl)porphine (TCPP), and 1,3,6,8-tetra(4-carboxylphenyl) pyrene(TBPy) by the solvothermal approach. The morphologies of Sm-TMA MOF, Sm-TCPP MOF, Sm-TBPy MOF were rod-shaped, cubic consisting of stacked 2D layers, and spherical made of small cubic structures, respectively. After the electrochemical properties of the synthesized MOFs were investigated, the MOFs were used to fabricate immunosensors for detection of carcinoembryonic antigen using a label-free signaling strategy. The immunosensors exhibited a wide linear detection range and a lower detection limit. The exhibited reproducibility and selectivity of the immunosensors were within the tolerable limits. The established label-free immunosensor has been successfully applied for detection of carcinoembryonic antigen in human serum samples, demonstrating that the rare earth metal-based MOFs are promising for construction of biosensors for medical diagnosis.

show abstract

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow

Cited by 17 publications

References 56 publications

Proteomes Are of Proteoforms: Embracing the Complexity

Proteomes Are of Proteoforms: Embracing the Complexity

Size-Based Proteome Fractionation through Polyacrylamide Gel Electrophoresis Combined with LC–FAIMS–MS for In-Depth Top-Down Proteomics

Morphologically Flex Sm-MOF Based Electrochemical Immunosensor for Ultrasensitive Detection of a Colon Cancer Biomarker

Contact Info

Product

Resources

About