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The recommended plasma or serum input volume is 20 µL. The minimum sample input volume is 10 µL, which might lead to a decrease in the protein identification rate. The maximum sample input volume is 40 µL; however, no dramatic increase in protein identification is observed by increasing the input volume.

With ENRICH-iST, approximately 10 µg peptide can be obtained from 20 µL of plasma/serum.

Yes, the ENRICH-iST workflow can be automated on liquid handling systems compatible  with magnetic bead handling and can be coupled with a positive pressure unit. We automated ENRICH-iST workflow on Tecan Fluent® Automation Workstation with Resolvex® A200. A corresponding poster is available here (A standardized, high-throughput platform for automated, rapid, and extensive plasma proteome characterization). We also partially automated the ENRICH-iST protocol using the Opentrons OT-2 system. Scripts and quick start guides can be shared upon request. For further information, please contact us at info@preomics.com.

The protein binding at 30°C is set for the most robust performance of ENRICH-iST. We do not recommend protein binding at RT as the temperature can vary from lab to lab or daily. During ENRICH-iST validation, the binding step was tested from 4 to 37°C without significant changes in the number of proteins identified, but it can affect the kit robustness.

The performance study did not cover washing beads in batches and distributing them afterward, but altered results are not to be expected.

This will vary depending on the LC-MS hardware used. We generally load 300 ng of peptides on an EASY-nLC™ 1200 system (Thermo Fisher Scientific) coupled to a timsTOF Pro/HT (Bruker); peptides are separated on a self-packed 30 cm column using a 25 min gradient and acquired using data-independent acquisition.

ENRICH-iST technology is based on the physicochemical binding of proteins to the beads in a specifically designed buffer. Further discloser of the ENRICH-iST methodology cannot be shared due to proprietary information.

Yes, ENRICH-iST can be used across multiple species, such as humans, mice, rats, etc.

Pre-treatments that induce conformation or structural changes in proteins affect the efficiency of protein enrichment on EN-beads.  Agents or workflows/treatments that denature proteins, such as heat exposure, protein precipitation, or the use of denaturing detergents are incompatible with ENRICH-iST.

ENRICHplus is offered as a 12x test size format and a 96x HT kit solution.

ENRICHplus 96x HT can be split into 4x 24 reactions.

The recommended plasma input volume is 50 µL. The minimum sample input volume is 20 µL, which might lead to a decrease in the protein identification rate. The maximum recommended sample input volume is 80 µL; however, no dramatic increase in protein identification is observed by increasing the input volume.

With ENRICHplus, approximately 3-4 µg peptide can be obtained from 50 µL of plasma.

ENRICHplus workflow can be automated on liquid handling systems compatible with magnetic bead handling and can be coupled with a positive pressure unit. We combined ENRICHplus workflow on KingFisher^TM Flex with eighter plate centrifugation for the clean-up part or with Tecan Fluent^® Automation Workstation Resolvex^® A200. ENRICHplus can be executed manually, semi-automated, or fully automated. Scripts and quick start guides can be shared upon request. For further information, please contact us at info@preomics.com.

Most peptide samples can be cleaned up with the washing buffers in the PreOmics’ kits. However, for certain samples such as urine or plant tissues, our regular iST or iST-NHS protocols can be extended with one additional washing buffer called “WASH0” to remove metabolites. When preparing peptide samples with high levels of contamination, e.g. sugars, fat, polymers or high concentrations of detergents, we recommend to use our PHOENIX peptide cleanup kit instead which  contains an additional wash step with “WASHX”.

The Phoenix peptide cleanup kit is able to remove detergents, sugars, lipids, salts and various polymers. For more details see our Phoenix Application Note.

You can either stack the 96-well CARTRIDGE adapter plate on top of the provided collection plate,or you can stack it on top of standardized autosampler vials. To avoid damage to the collection plate wells during the centrifugation step, use a suitable plate carrier together with the swing-bucket rotor (e.g. PCR plate adapter Eppendorf #5825711009 for Eppendorf plate rotors).

Concentrate at 45°C until the sample is dry and no residual ELUTE buffer is left. This usually takes about 30 min. Depending on your sample, peptide ions might accumulate at the top layer and thus interfere with efficient evaporation. To overcome this, tap the sample briefly to mix the eluate and then continue to concentrate in the vacuum concentrator.

Since our ELUTE buffer has a basic pH and C18 eluates have an acidic pH, do not place them in the same vacuum concentrator as this can damage the instrument.

Peptide quantification should be done in our LC-LOAD buffer and not in the ELUTE buffer. We recommend quantitative colorimetric peptide assays.

Storage of peptides should not exceed two weeks at -20°C. For long-term storage, keep them at -80°C.

CARTRIDGES in the iST and iST-NHS kits are the same. CARTRIDGES in the iST-BCT and PHOENIX kits are of different composition and have a slightly lower affinity for hydrophobic species.

It is essential to acidify the peptides, otherwise your sample will not bind to the CARTRIDGE. To do so, mix your peptides 1:1 with the provided STOP buffer and load everything on the CARTRIDGE.

Spin at 3,800 rcf for 1-3 min to load the sample completely.

Note: pH of STOP itself cannot be determined by pH measurement device or pH paper due to a high concentration of organic solvent. After adding 100 µL STOP to the sample,  pH can be measured by pH paper.

The iST-Fractionation Add-on kit contains three buffers, please note it does NOT contain the CARTRIDGE or LC-LOAD reagent, therefore this kit should ALWAYS be used in conjunction with one of the iST family of kits.

Yes, the iST-Fractionation Add-on kit is compatible with all of the PreOmics iST family of kits.

Add LC-LOAD to COLLECTION tubes 1-3, typically we suggest that you aim for 1 g/L concentration remembering that the starting protein concentration will have been fractionated into 3 (e.g. 90 µLto 90 µg protein starting material equates to 30 µL per tube).

Yes, all our kits are compatible with downstream peptide fractionation workflows. After peptides are dried completely, resuspend the peptides either in LC-LOAD or a resuspension buffer of your choice as input buffer for further peptide fractionation methods.

The SP3-iST Add-on kit contains SP3 beads and reagents for bead preparation, non-biased protein binding, lysis and washing. The kit must be used in conjunction with one of the iST, iST-NHS or iST-BCT kits.

Hughes, C.S., Moggridge, S., Müller, T. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14, 68–85 (2019). https://doi.org/10.1038/s41596-018-0082-x

For urea-containing buffers: To reduce the risk of carbamylation of proteins, it is recommended not to heat samples above 37 °C in urea-containing buffers. Please contact us via info@preomics.com for recommendations on how to modify the protocol.

Prepare your samples in your own extraction buffer (maximum volume 50 µL) and use the SP3 protocol as given, ensuring in step 2.1 that you add 50 µL of SP3 LYSE and make up to 100 µL with RESUSPEND if needed.

You do not have to use a combination of buffers as the protocol will work successfully using SP3 LYSE alone. Add 50 µl of SP3 LYSE to your sample and make up to 100 µl with RESUSPEND.

The SP3 LYSE buffer contains detergent and reduction agents needed. The alkylation reagent is added in the appropriate kit LYSE reagent.

The SP3 LYSE is not compatible with the BCA assay, we recommend starting with your usual extraction buffer and protein assay, if you do not have a preferred assay then we suggest the Detergent Compatible Bradford Assay from ThermoFisher https://www.thermofisher.com/de/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/bradford-assays.html

The bead volume is calculated from the number of samples being prepared and the protein concentration of those samples.For each sample starting protein concentration see the SP3 volume below:

E.g. for 3 samples containing 50 µg protein, transfer 3x 50 µL of SP3 BEADS

Yes, an aqueous elution step can be performed and may be useful when using the iST-BCT kit. Adjust the pH of sample to pH 8-9 with NaOH solution (added volume should not exceed 10 µL) and shake sample (RT; 1400 rpm; 5 min), then place the sample on the magnetic separator and remove the supernatant to a clean tube for the DIGEST step.

IMPORTANT: After the addition of STOP, make sure that the pH of the sample is acidic (pH 3-4).

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We offer a full pre-installation requirement documentation upon request. Briefly, the PreON operates at: 100–240 V AC, 50/60 Hz, 650 VA. PreON dimensions are (WxDxH): 65 cm (25.6 in.) x 62 cm (24.4 in.) x 86 cm (34.0 in.) with a weight of 71.5 kg (157.6 lbs).

The PreON can process protein cell and model organism pellets, body fluids and tissue lysates.

The PreON sample throughput is 12 label-free samples per run, or 16 samples in conjunction with TMTpro labeling reagents. An optional upgrade is also available, allowing PreON to run up to 18 samples by using TMTpro 18 plex labeling reagents. For iST label-free workflows, up to three runs per working day are feasible, to process in total up to 36 samples. For iST-NHS workflows, up to two runs per day are possible, for a total of 32  samples (36 samples if the TMT18plex upgrade is installed).

Yes, the PreON can execute workflows for both label-free and chemical labeling samples with our ready-to-go iST, iST-BCT and iST-NHS kits.

PreOmics recommends the use of  1.5 mL Lo-Bind Eppendorf tubes (Catalog No. 0030108442). Tubes that are not the correct dimensions may cause pipetting errors.

The PreON works seamlessly with our ready-to-go iST, iST-BCT and iST-NHS kits. Other methods or commercial products cannot be used with the PreON.

The PreON requires minimal maintenance such as cleaning the surfaces and emptying the solid and liquid waste containers. More detailed instructions can be found in the PreON software user interface.

Our trusted partner OneService is a leading global provider of technical services for medical, analytical and industrial equipment. Contact us to inquire about further servicing options and pricing.

PreON on-board lysis is the default setting when selecting the following sample types: Pellet, Liquid concentrated, or Liquid diluted.    

PreON on-board lysis consists of the following steps:

- Load PreON workdeck including your samples according to the instructions in the menu.

- LYSE buffer is added to the sample.

- Samples are heated (83 °C, 10 min, shaking) to effectively extract, denature and reduce and alkylate proteins.  

- NOTE: If you expect your samples to be viscous due to DNA released from the nuclei, nucleases can be used to reduce the viscosity of the samples, such as Benzonase. For more information and recommendations on the use of nuclease, see FAQ 1.25.  

PreON off-board lysis should be selected in the PreON menu when:

- LYSE buffer (50 µL) has already been added to samples manually.

- Working with samples that require a different lysis temperature (PreON lysis temperature: 83 °C).

- Working with samples that require mechanical homogenization/lysis, such as rigid tissue samples.

Differences between on-board and off-board lysis on the PreON:

For the PreON on-board lysis, the samples are incubated for 10 min at 83 °C with shaking. Heating the shaker to 83 °C and cooling it down to 37 °C for subsequent digestion takes an additionally ~15 min and ~25 min, respectively. The total time for the PreON on-board lysis step is therefore ~50 min.

Based on our use and testing, OS is very stable and can be used for up to 2 weeks. To ensure optimal performance, we recommend cleaning the OS bottle thoroughly each time before refilling it with fresh OS.

a. iST reagents (incl. resuspended iST-DIGEST) can be kept on deck if the ‘cooling to 10°C’ is active or be stored in the fridge for up to 5 days.

b. Resuspended iST-BCT DIGEST should be used as soon as possible. Avoid storing on the deck or in the fridge for over 12 hours.

The APP96 employs the same needle for multiple sample preparation steps; however, it includes several thorough washing steps for the needle, both inside and outside, after each sample handling step. Cross-contamination between samples has been thoroughly tested, and no contamination was detected.

a.       Yes, the SAMPLE PLATE can be reused across multiple days. For example, you can use positions 1 to 12 on Day 1 and positions 13 to 30 on Day 2.

b.       The plate foil can also be reused. If you prefer not to cut or reuse the foil, additional foils can be ordered  here:

One reagent bottle contains enough reagent to process up to 48 samples. However, processing smaller batches may require additional reagent due to dead volumes. We recommend using each reagent bottle for no more than 6 runs (e.g., 8 samples/run, totaling 48 samples).