Pressure-assisted sample preparation for proteomic analysis

Olszowy, Pawel; Burns, Ariel; Ciborowski, Paweł

doi:10.1016/j.ab.2013.03.023

Cited by 30 publications

(25 citation statements)

References 17 publications

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“…However, to include mixed Cys34 disulfides in the assay and to prevent unwanted reactions involving TCEP 18,19 , we investigated tryptic digestion without prior reduction of disulfide bonds. In fact, we observed that digestion of HSA with pressure cycling proceeded favorably without reduction of disulfide bonds, as reported previously 20 , and was enhanced with use of methanol to purify HSA. Treatment of HSA with 50% methanol – as used in the purification step – reportedly unfolds the ternary structure while maintaining the protein in solution 21 .…”

Section: Resultssupporting

confidence: 86%

Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin

et al. 2016

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An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a ‘housekeeping peptide’, and annotates modifications based on a combination of retention time, accurate mass, elemental composition and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g. ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.

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Section: Resultssupporting

confidence: 86%

Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin

et al. 2016

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“…Proteomic analysis of small amount of samples with a high level of reproducibility and quantitative accuracy remains technically challenging. We have recently developed an integrative methodology, PCT‐SWATH , comprising pressure cycling technology (PCT) for tissue lysis, protein extraction, and protein digestion, and Sequential Window Acquisition of all Theoretical Fragment‐Ion (SWATH)‐mass spectrometry (MS) for digitization of the proteome . From the resulting, highly convoluted fragment ion maps, proteins can be identified and quantified via a targeted data analysis strategy .…”

Section: Introductionmentioning

confidence: 99%

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT-SWATH mass spectrometry

et al. 2015

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The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH-MS to perform reproducible proteomic quantification of biopsy-level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT-SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3-0.2 mg). The data show that as few as 50 000 human cells and 0.2-0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH-MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT-SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.

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“…Recently, it has been shown that elevated hydrostatic pressure can improve protein extraction and reduce the digestion time of proteins from complex biological samples (Tao et al, 2007;Yang et al, 2010;Freeman & Ivanov, 2011;Olszowy, Burns, & Ciborowski, 2013). This type of technology was shown to be suitable for protein extraction from biopsy-sized tissues and is known as a pressure-cycling technology (PCT).…”

Section: B Pressure-based Systemsmentioning

confidence: 99%

Proteome analysis of tissues by mass spectrometry

Đapić

Baljeu‐Neuman

Uwugiaren

et al. 2019

Mass Spectrometry Reviews

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Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh‐frozen and formalin‐fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue‐sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom‐up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys‐C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue‐specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.

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Pressure-assisted sample preparation for proteomic analysis

Cited by 30 publications

References 17 publications

Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin

Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT-SWATH mass spectrometry

Proteome analysis of tissues by mass spectrometry

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