Proteomics Sample Preparation FAQ

How should proteins be preserved during sample collection to avoid degradation?

One effective strategy is to use ice-cold conditions during sample collection. Keeping samples on ice slows down enzymatic reactions that could lead to protein degradation. Additionally, using anticoagulants or protease inhibitors in the collection tubes can help inhibit the activity of enzymes that might degrade proteins. Another key factor is the timing of the collection and processing. Samples should be processed as quickly as possible to prevent any degradation that may occur during transport or storage. If immediate processing isn’t feasible, samples can be snap-frozen in liquid nitrogen or stored at ultra-low temperatures (-80°C) to maintain protein integrity.

How does the choice of anticoagulants during blood sample collection affect proteomics results?

The choice of anticoagulants during blood sample collection can significantly influence proteomics results, as different anticoagulants can affect protein stability and activity. Common anticoagulants like EDTA and heparin have distinct effects on the coagulation cascade and can alter the proteome in blood samples. For instance, EDTA is a chelating agent that binds calcium ions, preventing coagulation and preserving plasma proteins but may also interfere with metal-dependent enzymes, potentially skewing results. Moreover, some anticoagulants may introduce their own contaminants or inhibit certain assays used in downstream proteomics analysis. For example, citrate anticoagulants may affect the solubility of certain proteins, impacting their detection in mass spectrometry. Therefore, it's essential to select an anticoagulant that best preserves the proteins of interest and aligns with the goals of the study.

How does freeze-thawing affect protein integrity in collected samples?

Freeze-thawing can significantly impact protein integrity, leading to degradation or denaturation. When a sample is frozen, ice crystals form, which can disrupt cellular structures and potentially denature proteins. Repeated freeze-thaw cycles can exacerbate this effect, causing additional stress that might alter protein conformation or functionality. For example, enzymes can become inactive, and some proteins may aggregate, making them less detectable in downstream analyses like mass spectrometry. To minimize these effects, it's recommended to aliquot samples into smaller volumes before freezing, so only a portion is thawed at a time. This way, the bulk of the sample remains frozen, preserving protein integrity. Additionally, using cryoprotectants, such as glycerol or trehalose, can help protect proteins during freezing and thawing.

How does fasting or diet influence the protein profile in collected samples?

Fasting or diet can have a significant impact on the protein profile in collected samples, as these factors influence metabolic processes and the overall composition of biological fluids. For instance, a high-fat meal can temporarily elevate specific proteins involved in lipid metabolism, while fasting might lead to changes in proteins related to energy metabolism or stress responses. This can affect the interpretation of results, especially in clinical studies where dietary habits may vary among subjects. When conducting proteomic studies, it's important to standardize fasting periods or dietary conditions for all participants to minimize variability. We should also consider documenting participants' dietary habits leading up to sample collection. This information can provide context for any observed changes in protein profiles and help in the accurate interpretation of data, especially in studies related to metabolic diseases or nutritional impacts.

How much sample volume is required for MS-based proteomics analysis?

The required sample volume for mass spectrometry (MS)-based proteomics analysis can vary depending on several factors, including the sensitivity of the method, the type of sample, and the specific proteins of interest. Generally, for proteomics studies using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a typical starting volume is around 100 to 200 microliters of plasma or serum. However, for tissue samples, a larger volume may be needed, especially if the protein concentration is low. It's crucial to optimize the sample volume based on the specific assay and instrument sensitivity. For low-abundance proteins, researchers might require larger volumes or highly concentrated extracts to ensure sufficient protein detection.

Can you use the same protocol for protein collection from different species or tissues?

While it's tempting to apply the same protein collection protocol across different species or tissues, this approach may not always yield optimal results. Different species can have varying protein compositions and structures, and their tissues may respond differently to extraction methods. For example, a protocol that works well for mammalian tissues might not be suitable for plant tissues due to differences in cell wall composition and the presence of unique metabolites. Additionally, specific tissues within the same species may require tailored approaches. For instance, extracting proteins from muscle tissue might necessitate a different lysis buffer compared to brain tissue, as they have distinct cellular environments. For us, we optimize extraction protocols for each specific tissue type or species to achieve the best protein yield and quality, ensuring accurate downstream analysis.

How should plant samples be collected for proteomics studies?

It’s essential to collect samples at the appropriate time of day, typically in the morning when plants are less stressed and have higher metabolic activity. Sampling during dry conditions can also help minimize moisture-related degradation of proteins. Using clean, sharp tools to cut samples reduces damage and contamination. Once collected, plant tissues should be quickly frozen in liquid nitrogen to halt enzymatic activity and preserve protein integrity. For some studies, it may be beneficial to grind the frozen tissue in the presence of liquid nitrogen to ensure a homogeneous powder for extraction. Additionally, choosing the right extraction buffer that suits the specific plant tissue is crucial for optimal protein yield.

How does circadian rhythm affect protein expression in time-sensitive sample collection?

Proteins can exhibit diurnal patterns, meaning their levels fluctuate depending on the time of day. For example, cortisol and certain clock proteins display distinct expression patterns that align with the light-dark cycle. This variability can affect the outcome of proteomics studies if samples are not collected at consistent times. To account for these circadian influences, we should standardize sample collection times relative to the light cycle, ideally at the same time each day across all subjects. Documenting the time of collection can provide valuable context for interpreting protein expression levels.

What is the best way to collect cell culture samples for proteomics analysis?

Collecting cell culture samples for proteomics analysis requires careful handling to ensure protein integrity and quality. First, it's essential to choose the right time point for collection, often after a specific treatment or under specific conditions relevant to the study. Before collection, researchers should wash the cells gently with a buffer, such as phosphate-buffered saline (PBS), to remove any serum proteins or media components that could interfere with the analysis. After washing, cells can be lysed directly in the culture dish using an appropriate lysis buffer containing protease inhibitors to prevent protein degradation. The lysate should then be collected and promptly frozen or processed for analysis. For adherent cells, scraping can be an effective way to collect cells, while for suspension cultures, centrifugation followed by resuspension in lysis buffer works well.

How should fecal samples be collected for protein analysis in microbiome studies?

Collecting fecal samples for protein analysis in microbiome studies requires specific considerations to ensure sample integrity and avoid contamination. First, we should provide participants with sterile collection containers to minimize external contamination. It’s best to collect fresh samples to capture the most accurate protein profiles, as prolonged storage can lead to protein degradation or changes in microbial composition. After collection, fecal samples should be processed as quickly as possible, ideally within a few hours. For proteomic analysis, we may choose to freeze the samples immediately in liquid nitrogen or store them at -80°C to preserve protein integrity. Additionally, it's important to homogenize the samples effectively to ensure a representative aliquot for analysis.

How to prevent protein oxidation during sample collection?

Oxidation can lead to changes in protein structure, function, and ultimately, misinterpretation of data in proteomics studies. To mitigate oxidation, we should perform all sample handling steps in an environment with minimal exposure to oxygen. This can be achieved by working under anaerobic conditions or using inert gases, like nitrogen, to displace oxygen in the sample environment. Additionally, incorporating antioxidants into lysis buffers or sample containers can further protect proteins from oxidative damage. Common antioxidants include dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), which can help maintain reduced protein states.

Are there special considerations for collecting protein samples for phosphoproteomics?

Yes, collecting protein samples for phosphoproteomics involves special considerations due to the dynamic nature of phosphorylation and the sensitivity of phosphoproteins to degradation. It’s crucial to use lysis buffers that contain phosphatase inhibitors, as these enzymes can rapidly remove phosphate groups from proteins during sample processing, leading to loss of critical information regarding phosphorylation status. Timing is also essential; samples should be collected quickly to minimize changes in phosphorylation states. In some cases, researchers might choose to flash-freeze samples in liquid nitrogen immediately after collection to halt enzymatic activity. Furthermore, when analyzing phosphoproteins, it’s advisable to use enrichment techniques, such as immunoprecipitation or specific chemical methods, to isolate phosphorylated proteins effectively.

We are interested in DIA proteomics using human neuronal cells. The cells count will be about 0.5 million. Is this sufficient for you?

At least 1 million cells are required for proteomics analysis.

Can we do whole blood for proteomics? Or only plasma or serum?

we can process whole blood samples for proteomics.

I am interested in protein profiling using cultured cells or human plasma. Does your DIA/DDA or blood proteomics cover any proteins we want to focus?

Yes, we can. I checked these proteins that are mentioned and found they can be identified in our blood proteomics.

I'd like to add proteomics to our transcriptomes data for a phage (viral) infection of bacterial life cycle. Do you have any experiences working with bacterial proteomics?

Our proteomics services accommodate microorganism samples. For bacteria, a minimum of 100 mg is required, with 200 mg recommended for optimal results. For fungi, a minimum of 150 mg is needed, with a recommendation of 300 mg for best outcomes.

We want to do DIA proteomics, and would like to submit protein samples. The samples has been desalted and ready to be injected. Can we take this and inject it directly for DIA? The protein sample is in the following format: the buffer with the samples: 2% ACN/H2O (0.1% FA) containing iRT (50ng:100ug protein)

Yes, we can work with these samples. Based on the buffer information, the samples should be peptides. Therefore, the recommended sample size is 20 µg, with a minimum sample size of 10 µg.

I am interested in doing Phosphoproteomics. Our samples are mouse brain tissues (cortex and hippocampus). The protein is a membrane protein on the mitochondria. Do you need any speical extraction in order for us to better capture these type of proteins?

We recommend that you separate the mitochondria from mouse brain tissue using kits or other available methods, as we do not offer this specific assay. Please use at least 200 mg of tissue to isolate the mitochondria and then send the mitochondrial samples to us. We will extract the proteins from the mitochondria using personalized methods with RIPA lysis buffer.

I am interested in submitting EV samples from human plasma. Considering that these EVs are derived from plasma, will the DIA Proteomics panel or would the Serum/Plasma Proteomics panel work better?

DIA is suitable. Serum/Plasma Proteomics panel only works with serum or plasma samples and there is a enrichment step to enrich the low abundance proteins in blood. If the sample is EVs separated from plasma, there is no need to do the enrichment.

We wants to submit exosome samples from donkey milk to run DIA proteomics. What is the sample requirement? I saw on the website it says exosome (sediment) 25ul recommended, 15ul minimum.We also wants to do metabolomics and lipidomics. What is our requirement for metabolomics? Is it still by volume? Or is it the same as the EV particle number requirement?

Yes, for exosomes prepared for metabolomics detection, the sample requirements are the same as those for EVs: 2*10^9 particles (NTA) are recommended, with a minimum of 1*10^9 particles (NTA). For exosomes prepared for DIA/DDA proteomics detection, the required sample size is 1*10^11 particles (NTA) recommended, with a minimum of 5*10^10 particles (NTA). The recommended volume of exosome sediment is 25 µl, with a minimum of 15 µl being acceptable.

I am interested in doing proteomics for whole blood. Can whole blood be used for our serum/plasma proteomics?

Whether you want to detect proteins within the blood cells or to remove the blood cells before proteomics detection? Depending on the requirement, different pre-processing methods should be taken to the whole blood samples.

I am interested in our DIA Quantitative Proteomics platform. Do you have experience with urine samples for this platform, specifically with extracellular vesicles? Additionally, if we can filter out cellular and bacterial protein in urine for this platform.

We have experience with DIA proteomics on both urine and extracellular vesicle (EV) samples, though not specifically on EVs isolated from urine. For urine samples, our pre-processing includes a centrifugation step to help remove cells or bacteria. However, we recommend that clients purify their urine samples themselves, such as by passing them through an ultrafiltration tube, to ensure the removal of cells or bacteria. Regarding EV samples, we only process well-prepared EVs, which clients are responsible for extracting themselves.

I am interested in proteomics for stool samples. Is this something we can do?

We can process stool samples using our DIA proteomics, with a minimum required sample size of 500 mg. Please note that this method is not metaproteomics.

For DIA proteomics: Can you detect proteins with chemical adducts attached? Can you detect any phosphorylation sites in DIA? Or if we do phosphoproteomics, are you only going to detect phosphorylation, or are any regular proteins in DIA that can also be detected in phosphoproteomics?

1). In DIA proteomics, we cannot detect proteins with attached adducts. If you wish to identify these proteins, DDA proteomics is a suitable alternative. The modified position and information about the adduct should be provided. After LC-MS detection, we can perform a custom database search to identify proteins with specific chemical adducts. 2). Using DIA proteomics, we cannot detect phosphorylation sites. However, in phosphoproteomics, due to the enrichment step, over 90% of the detected proteins are phosphorylated. Some regular proteins may also be detected and will be included in the protein list provided in the final deliverable to clients.

We have precipitated peptides (PP) from cell free supernatant and those molecules that could be both larger and smaller weight proteins and test for inhibitory action. PP gave even better activity in vitro. Are we able to detect precipitated peptides? Could your proteomics help?

We can identify and relatively quantify the precipitated peptides using our proteomics services. However, for precipitated peptide samples, we will only desalt and run the LC-MS without performing the digestion step. Therefore, if the sample contains a mixture of peptides and proteins, we will not be able to detect the proteins in the sample.

We are interested in our QL and DIA proteomics. We works with mice mast cell lines. Can you please clarify the sample requirement for this specific type of cells? Any recommendations in sample preparation?

Mast cell lines can be treated like normal animal cell lines for sample requirements and preparation. For the QL panel, a minimum of 10^6 cells per sample is required, with 10^7 cells recommended. For the DIA proteomics panel, at least 10^6 cells per sample are needed, with 2*10^6 cells recommended. Please refer to the following figure for sample preparation suggestions.

I am interested in doing DIA Proteomics on Trabecular Meshwork and Retinal Ganglionic cells. Do your have any specific recommendation on these cell types?

No special requirements are required for these cell types. For sample requirements related to cells, regular cells can follow the cell count guidelines outlined in our sample requirement form. If the cell size is significantly larger or smaller, collecting at least 20 µL of cell pellet is acceptable.

Do you have experience doing DIA proteomics on cow milk? What's your past detection rate in terms of number of protein detected? Is 20ul sufficient?

We had several DIA projects on the EVs extracted from milk. The number of detected proteins is up to 8000. 20 ul of milk is acceptable to do DIA proteomics.

Can you provide a demo dataset for human cell samples running DIA Proteomics?

Yes, we can provide a deomo report.

I am interested in doing Quantitative Lipidomics and DIA proteomics on human brain tissue. Do we have a demo dataset for each service working with human brain tissues? Do we have any experiences working with post-mortem brain tissue samples?

Yes, we have demo datasets for Quantitative Lipidomics and DIA proteomics on human brain tissue for you.

We want to do DIA proteomics on dehydrated placenta tissue. The key question is we are interested in concentration values of the quantified protein. Is this something you can help with from our workflow?

Our quantitative proteomics services can provide relative quantification of proteins. However, absolute quantification is not available through these services.

I am interested in our phosphoproteomics panel. We work with fatty tissue samples. How much material do we need for phosphoproteomics? Is it 500mg - 1g of fatty tissue? The fatty tissue is highly abundant in lipids. Does that have any interference with our phosphoproteomics workflow?

500mg - 1g of fatty tissue would be adequate for the analysis. We will perform specialized preprocessing on the fatty tissues to address the high lipid content before conducting phosphoproteomics analyses.

For DIA proteomics, is Mitochondrial Respiratory Chain Enzyme Analysis included as part of our standard data analysis when working with cell pallets? Or is this something else we can help when analyzing the data?

No, DIA proteomics analysis extracts and detects all proteins in a given sample. Mitochondrial Respiratory Chain Enzyme Analysis is not something that DIA covers. Unfortunately, we do not currently offer Mitochondrial Respiratory Chain Enzyme Analysis services.

We did not list CSF needing high abundance protein depletion in our proteomics brochures, but they did say Albumin is the most abundant protein in CSF and we should also do depeltion for CSF as well.

Our past experience has shown that up to 9,000+ proteins can be detected in CSF samples without depleting high-abundance proteins. This demonstrates that high-abundance proteins in CSF do not significantly impact proteomics analysis. Moreover, both high-abundance protein depletion and low-abundance protein enrichment can alter the original protein profile in the samples. Therefore, unless necessary, we do not recommend these preprocessing steps.

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