Proteomics
Shotgun proteomics are bottom-up proteomic techniques to identify proteins in complex mixture with mass spectrometry.
Multiple fractionation or separation methods are often combined to improve signal-to-noise and proteome coverage and to reduce interference between peptides in quantitative proteomics. Furthermore, a given fractionation method provides additional information on the analytes, such as molecular weight, hydrophobicity or isoelectric point that can be used to improve identification, and to discover protein splice variants or large post-translational modifications.
After cell lysis or isolation, lysates often contain contaminants that are not compatible with downstream steps.
AFFINISEP has developed special tools based on AttractSPE®Disks that can use simply and efficiently for:
- Desalting :
Before MS analysis of peptides or proteins, the desalting step is a crucial step to remove salts and purify the peptides/proteins from in-gel and in-solution digests.
The choice of the sorbent is important as the sorbent will bind peptides and allow salt and other contaminants to be washed off the sample. Then clean peptides and proteins are eluted and analyzed. - Fractionation:
Peptide fractionation is an extremely important and challenging step in proteomics approaches facing more and more complex mixtures.
The goal is to simplify each fraction for a simplified analysis to extract more information. - Detergent removal:
For a good solubilization of proteins and their digestions, detergents are required.
These compounds can then affect the peptides/proteins identification and their removal is an important step for greater peptide identification. - Exchange buffer.
Several sorbents can be used such as HLB, C18, C8, DVB (hydrophobic), ionic polymers for weak or strong interactions (RPS,SAX, WAX, SCX, WCX)… The product exists under various formats from Stage tips (SPE tips), Spin SPE, 96 Plates SPE (1mL or for microelution) or SPE columns based on disks. If you need to scale-up your purification, the exactly same resin is available under cartridge format.
Several formats are available depending on sample size : SPE tips, 2mL/15mL/50mL spin tubes, cartridges with SPE Disks or 1mL/microelution 96 well plates.
To address the need for high throughput and rapid removal of small molecules from protein sample preparations, we have expanded our offering with 96 SPE Plates.
Our Biomolecular applications booklet collects main useful information about :
- Spinnable, Automatable, High throughput HT, Micro-SPE for Microextraction
- Proteomics
- Biomarker discoveries
- Biological applications
> See our Biomolecular applications booklet.
- Proteomics
Poster Development and optimization of a method for automated peptide desalting
New!
- Proteomics
Estimation of the working range on BioSPE™PurePep SPE Tips – Standard capacity
New !
- Proteomics
Affinisep poster BioSPE™ PepFrac – Fractionation
New poster!
- Proteomics
Identification of more than 97% proteins after purification by using the StageTips AttractSPE®Disks Tips C18
- Proteomics
AN AttractSPE®Disks Tips C18 - Peptides
The time we spend asleep is adjusted to previous time spent awake, and therefore believed to be under tight homeostatic control. By using quantitative phosphoproteomics, authors demonstrate that microgliaderived TNFα controls thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep-promoting kinases and numerous synaptic proteins, including a subset whose phosphorylation status codes sleep need and determines sleep duration. As a result, lack of microglial TNFα attenuates the build-up of sleep need, as measured by slow wave activity, and prevents immediate compensation for loss of sleep.
Desalting of tryptic digest was performed with home-made StageTips, made by stacking three layers of AttractSPE®Disks Bio C18 (SPE-Disks-Bio-C18-47.20) into 200µL pipette tips (equivalent to our AttractSPE® Disks Tips C18, ref Tips-C18.T3.200.96, and containing 2mg of C18 material).
New ! Microglial TNFα orchestrates brain phosphorylation during the sleep period and controls homeostatic sleep, Maria J Pinto, Léa Cottin, Florent Dingli, Victor Laigle, Luís F. Ribeiro, Antoine Triller, Fiona J Henderson, Damarys Loew, Véronique Fabre and Alain Bessis, 2022. OPEN ACCESS.
Aging affects iron homeostasis, as evidenced by tissue iron loading and toxicity and common anemia in the elderly. Iron needs in mammals are met primarily by iron-recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age dependent decline, a condition that has been largely unexplored to date. Results of this study showed that 10-11-months-old female mice exhibit iron loading, diminished lysosomal activity, and decreased erythrophagocytosis rate in RPMs.
Tryptic peptides were desalted with AttractSPE®Disks Bio C18 (SPE-Disks-Bio-C18-47.20) packed into StageTips (equivalent to our AttractSPE® Disks Tips C18, ref Tips-C18.T1.200.96).
New ! Impaired iron recycling from erythrocytes is an early hallmark of aging, Patryk Slusarczyk, Pratik Kumar Mandal, Gabriela Zurawska, Marta Niklewicz, Komal Kumari Chouhan, Matylda Macias, Aleksandra Szybinska, Magdalena Cybulska, Olga Krawczyk, Sylwia Herman, Michal Mikula, Remigiusz Serwa, Malgorzata Lenartowicz, Wojciech Pokrzywa and Katarzyna Mleczko-Sanecka, 2022. OPEN ACCESS.
Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of these proteins. Here, a series of analogues have been prepared and few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line.
In this study, stagetips based on AttractSPE®Disks Bio C18 (SPE-Disks-Bio-C18-47.20) and equivalent to our AttractSPE® Disks Tips C18 (ref Tips-C18.T1.200.96) were used to desalt peptide mixtures prior to LC-MS analysis.
New ! Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase, Joanna Małolepsza, Aleksandra Marchwicka, Remigiusz A. Serwa, Sanna P. Niinivehmas, Olli T. Pentikäinen, Edyta Gendaszewska-Darmach & Katarzyna M. Błażewska, Journal of Enzyme Inhibition and Medicinal Chemistry, Volume 37, Issue 1, 940-951, 2022. OPEN ACCESS.
Plant hormons cytokinin (CK) were purified with high efficiency thanks to the combination of a three-layered C18 – RPS – SCX Stagetips to a study to improve plant adaptation to heat stress.
New ! Priming Maritime Pine Megagametophytes during Somatic Embryogenesis Improved Plant Adaptation to Heat Stress; María Amparo Pérez-Oliver, Juan Gregorio Haro, Iva Pavlovic, Ondrej Novák, Juan Segura, Ester Sales, Isabel Arrillaga, Plants 2021, 10, 446. OPEN ACCESS.
Collagen peptide mass fingerprinting, best known as Zooarchaeology by Mass Spectrometry (or ZooMS) when applied to archaeology, has become invaluable for the taxonomic identification of archaeological collagenous materials, in particular fragmentary and modified bone remains. Prior to MALDI-based spectrometric analysis, collagen needs to be extracted from the bone’s inorganic matrix, isolated and purified.
Archaeological samples were purified according to SP3 (Single-Pot, Solid-Phase, Sample-Preperation)method using StageTips (equivalent to AttractSPE®Disks Tips C18 ref C18.T2.200.96) from two trimmed AttractSPE® Disks Bio C18 (SPE-Disks-Bio-C18-100.47.20).
New ! Testing the efficacy and comparability of ZooMS protocols on archaeological bone, Wang Naihui, Brown Samantha, Ditchfield Peter, Hebestreit Sandra, Kozilikin Maxim, Luu Sindy, Wedage Oshan, Grimaldi Stefano, Chazan Michael, Horwitz Kolska Liora, Spriggs Matthew, Summerhayes Glenn, Shunkov Michael, Richter Korzow Kristine, Douka Katerina, Journal of Proteomics, Volume 233, 104078, 2021.
Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. In this papers, researchers from several institutes of University of Cologne (Germany) have studied the role of Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability.
During there experiments, protein identification prior to mass spectrometry, AttractSPE®Disks Bio RPS (SPE-Disk-Bio-RPS-47.20) have been used as Stagetips.
New ! Ecd promotes U5 snRNP maturation and Prp8 stability, Steffen Erkelenz, Dimitrije Stanković, Juliane Mundorf, Tina Bresser, Ann-Katrin Claudius, Volker Boehm, Niels H Gehring, Mirka Uhlirova, Nucleic Acids Research, 2021; gkaa1274, OPEN ACCESS.
In this article, proximity labeling technique optimized for studying proteins in yeast mitochondria is described. Importantly, BioID (biotin identification) permits the identification of transiently interacting proteins and also proteins located in vicinity of the bait, which may not be co-purified by conventional pull down experiments. Stagetips based on AttractSPE®Disks Bio RPS (SPE-Disks-Bio-RPS-47.20) or commercially available (Tips-RPS-M.T2.200.96) were used to purify bionylated proteins prior to Mass spectroscopy analysis.
Mapping protein networks in yeast mitochondria using proximity-dependent biotin identification coupled to proteomics, Roger Salvatori, Wasim Aftab, Ignasi Forne, Axel Imhof, Martin Ott, Abeer Prakash Singh, STAR Protocols, Volume 1, Issue 3, 2020, 100219, OPEN ACCESS.
In this article, high-throughput genetic screens were performed that provide a novel global map of histone residues required for transcriptional reprogramming in response to heat and osmotic stress.
In this study, fractionation of phosphopeptides at high pH was performed using gel loading tips packed with AttractSPE®Disks C18 (equivalent to our AttractSPE® Disks Tips C18 and containing 1 mg C18 material.
A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress, Cristina Viéitez, Gerard Martínez-Cebrián, Carme Solé, René Bö ttcher, Clement M Potel, Mikhail M Savitski, Sara Onnebo, Marc Fabregat, Ali Shilatifard, Francesc Posas, Eul`lia de Nadal, Nucleic Acids Research, Band 48, Ausgabe 7, 17. April 2020, Seiten 3455–3475, OPEN ACCESS.
The combination of thermal proteome profiling (TPP) with phosphoproteomics (phospho-TPP) offers a way to assess the functional relevance of identified phosphorylation sites on a proteome-wide scale by comparing the melting behaviour of a protein and its phosphorylated form(s). In this article, phospho-TPP experiments were performed in HeLa cells using an optimised protocol.
In this study, phosphopeptide fractionation was performed with gel loading tips plugged with AttractSPE®Disks C18 (equivalent to our AttractSPE® Disks Tips C18 and packed with 1 mg C18 material.
Impact of phosphorylation on thermal stability of proteins, Clément M. Potel, Nils Kurzawa, Isabelle Becher, Athanasios Typas, André Mateus, Mikhail M. Savitski, bioRxiv 2020.01.14.903849; Open access.