A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

Padula, Matthew P.; Berry, Iain J.; Rourke, Matthew B O; Raymond, Benjamin B. A.; Santos, Jerran; Djordjevic, Steven P.

doi:10.3390/proteomes5020011

Cited by 43 publications

(29 citation statements)

References 203 publications

(248 reference statements)

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“…Altogether, these results suggest that optimising EF and lysis duration can significantly improve separation performance and protein solubilisation. Further improvements of solubilisation may be achievable via chemical modulation, as has previously been reported in conventional preparatory IEF, 73,74 or perhaps by modulating the substrate to allow for laser-based lift off of adherent cells 75 prior to lysis and IEF.…”

Section: Resultsmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

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Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

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Section: Resultsmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

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“…Using IPG strips, the first-dimension separations are more reproducible and have high throughput and high resolution. The IPGs are much easier to handle, and there is the convenience provided by commercial production of IPG strips made [33,35,36].…”

Section: Two-dimensional Gel Electrophoresis (2dge)mentioning

confidence: 99%

Proteoforms: General Concepts and Methodological Process for Identification

Araújo¹,

Machado²

2020

Proteoforms - Concept and Applications in Medical Sciences

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The term proteoform is used to denote all the molecular forms in which the protein product of a single gene can be found. The most frequent processes that lead to transcript modification and the biological implications of these changes observed in the final protein product will be discussed. Proteoforms arising from genetic variations, alternatively spliced RNA transcripts and post-translational modifications will be commented. This chapter will present an evolution of the techniques used to identify the proteoforms and the importance of this identification for understanding of biological processes. This chapter highlights the fundamental concepts in the field of top-down mass spectrometry (TDMS), and provides numerous examples for the use of knowledge obtained from the identification of proteoforms. The identification of mutant proteins is one of the emerging areas of proteogenomics and has the potential to recognize novel disease biomarkers and may point to useful targets for identification of therapeutic approaches.

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“…lipids and phospholipids) can cause ion suppression. To obtain better sensitivity and robustness, the usual approach is to remove high‐abundance matrix interferences by applying extraction procedures at the protein and/or peptide levels, such as immunodepletion, immunocapture, or solid‐phase extraction [26–32]. Immunoaffinity‐based sample pretreatment techniques are quite expensive, usually not suitable for multiplexed analysis, and there can be cross‐reactivity, as well as difficulties in the transfer of the methodology in‐between different laboratories.…”

Section: Introductionmentioning

confidence: 99%

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

Maráková

Alex

Schug

2020

J of Separation Science

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In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancerrelated intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30 • C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields. K E Y W O R D Sbiological fluids,

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A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

Cited by 43 publications

References 203 publications

Electrophoretic cytometry of adherent cells

Electrophoretic cytometry of adherent cells

Proteoforms: General Concepts and Methodological Process for Identification

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

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