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We recommend that you use the appropriate LYSE buffer provided with the PreOmics’ iST, iST-NHS or iST-BCT kits. If you want to use your own lysis buffer, please refer to the following table For all other buffers containing higher concentrations of salt, chaotropes, or detergents, we recommend using the SP3-iST Add-on kit in combination with the iST, iST-NHS, and iST-BCT kits (see FAQ 8.2 for more information on SP3-iST compatibility).

2X concentrated LYSE buffers have to be ordered in addition to the iST kits. Please contact us (info@preomics.com) for any further question on buffer compatibility or supply with reagents.

Sample volume (liquid or solid, e.g. cell pellets) should not exceed 10 µL when combined with the appropriate LYSE buffer. For sample volumes between 11-25 µL,use the 2-fold concentrated lysis buffers, which can be purchased upon request. The 2X lysis buffers are mixed 1:1 with samples ranging from 11-25 µL, e.g. 11 µL sample + 11 µL 2X LYSE,  all downstream buffer volumes stay the same.

If your sample volume exceeds 25 µL, perform protein precipitation before processing with the standard PreOmics’ workflows.

Several different protocols for protein precipitation exist. We recommend acetone precipitation:

1. Transfer protein lysate (not more than 300 µL) to a clean 2 mL microcentrifuge tube.
> in case you have a larger sample volume, use either a 5 or 15 mL tube
2. Add ice-cold acetone (-20°C) to your sample > add at least 4-fold more acetone than sample, e.g. 300 µL sample + 1.2 mL acetone
3. Mix briefly
4. Incubate for one hour at -20°C
5. Spin in table-top centrifuge at 4°C for 15 min at 13,000 rpm
6. Carefully discard supernatant, make sure not to disturb the pellet
7. Air-dry the pellet for 5-10 min
8. Continue with appropriate PreOmics protocol adding the LYSE
> you can also freeze the pellet after air-drying at -20°C or -80°C until further use

Although protease inhibitors are in general compatible with our iST protocols, we recommend not to add additional exogeneous proteins in order to not contaminate your sample.

Many different mechanical force methods can be employed such as traditional bead milling, liquid nitrogen grinding, or commercial systems from various vendors (e.g. Bertin Instruments, Covaris, Hielscher, MP Biomedicals). These methods can aid lysis efficiency for samples including cells, yeast, or tissues. We recommend the PreOmics' BeatBox system which is easy to use and allows efficient and reproducible tissue homogenization and cell lysis with flexible throughput from 1-96 samples.
See https://www.preomics.com/beatbox for more information.

Samples such as biological fluids do not require additional mechanical force disruption.

Highly reproducible results are achieved with protein starting amounts ranging from 1 µg to 100 µg.
For low input information please see "Shall I adjust the buffer volumes depending on the protein starting amounts?"

To determine protein quantity in the sample lysed in PreOmics LYSE buffers, we recommend using the Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible or the NanoOrange™ Protein Quantitation Kit (Thermo Fisher Scientific), depending on which LYSE is used. Below is the list of compatible LYSE buffers and the protein quantification assay with recommended dilution.

Protein quantification assays and compatible PreOmics LYSE buffers

To determine peptide quantity in the sample after using the PreOmics kit family, we recommend using the Pierce™ Quantitative Fluorometric, Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific). Below is the list of compatible PreOmics buffers and the peptide quantification assay with recommended dilution. Please note that peptide quantification in diluted ELUTE should only be used as a rough estimation. We recommended performing peptide quantification in the LC-LOAD buffer.

Peptide quantification assays and compatible PreOmics buffers

* Peptide quantification in diluted ELUTE should only be used as a rough estimation. We recommend doing the peptide quantification in LC-LOAD.
** For Fractionation workflow, we recommend Pierce colorimetric assay.

The maximum volume is 250 µL.

We recommend using the buffer volumes indicated in the protocol. You can adjust the volume of LYSE and DIGEST to your starting amounts. For less than 20 µg protein start-ing material use the following volumes: 10 µL LYSE; 10 µL DIGEST; keep the other buffers as indicated in the protocol. Accordingly, you may adjust the volumes for chemical labeling and quenching as recommended by the label manufacturer.

Perform the lysis/denaturation step at 95°C. Use lower temperatures only for temperature-sensitive samples such as immunoprecipitations. PreOmics have tested lower temperatures down to 60°C and do not see any differences in parameters assessed (e.g. IDs, alkylation rates).

The PreOmics’ kits come with a lyophilized enzyme mix consisting of LysC and trypsin, we do not supply other enzyme combinations.

The digestion depends on your sample type and input material (see table below). Please note our recommendations to lower the enzyme amount when working with low input samples (<20 µg protein starting material, see 1.9.)

Although not recommended, you can also digest your samples overnight (~18 hours). While this will reduce the missed cleavage rate even further, it will come at the cost of higher unspecific cleavages and much longer processing time of the overall workflow.

While our protocols are in general compatible with IP samples (modified protocols can be found to download on our website), the required input amounts for global phosphorylation enrichment experiments (~500 µg) usually exceed the peptide binding capacity of our CARTRIDGES.

The PreOmics' PreON isa dedicated sample preparation platform suitable for 12 or 16 samples, see here or click on the "PreON platform" tab for more information. PreOmics is working with liquid handling systems providers and application notes are available here or we are happy to provide technical assistance to transfer the PreOmics methodology onto your existing platform if appropriate.

All of the PreOmicskits are compatible with absolute quantification strategies. For absolute quantification employing isotopically-labeled protein standards, please add the respective absolute standard (e.g. DIGESTIF, PSAQ, SILAC-PrEST) together with your sample to the appropriate LYSE buffer ensuring the total protein does not exceed 100 µg and proceed with the protocol accordingly.

For absolute quantification employing isotopically-labeled peptide standards with the PreOmics' kits, please introduce the respective standard (e.g. AQUA, QconCAT) before adding the STOP buffer.

The best way to assess peptide recovery rates is by employing absolute quantification strategies (see 1.15). Our peptide recovery is typically >80% for 1 - 100 µg protein starting material.

Production of PreOmics’ kits is ISO 9001:2015 certified. Quality control measures include incoming goods control, polymer leaching tests for plasticware, LC/MS-based quality control for parameters including alkylation rate or missed cleavages on a standardized sample. Upon request, PreOmics can provide a certificate of analysis (CoA) to our clients.

Our kits are provided in pack sizes according to the number of samples that can be processed:

iST kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs

iST-REG-PSI kits
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs, includes 2 fixed well plates for the PURIFY step.

iST-NHS kits
◦ 4 reactions: test kits for first time users
◦ 12 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes
◦ 192 reactions: large kit suitable for liquid handling platforms, supplied in 20 mL vials for convenient transfer into liquid handling reservoirs

iST-BCT kits
◦ 4 reactions: test kits for first time users
◦ 8 reactions: small kits for initial experiments
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers and enzymes are supplied in 2 mL tubes

Phoenix kits
◦ 4 reactions: test kits for first time users
◦ 96 reactions: medium kits with flexibility to process either up to 96 samples at once or multiple smaller batches, as all buffers supplied in 2 mL tubes

Yes, all our kits are compatible with downstream peptide fractionation workflows. PreOmics has a dedicated iST-Fractionation Add-on kit or alternatively 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 iST-BCT kit has been designed specifically for use with biological fluids plasma and serum samples and for analysis of monoclonal antibodies, therapeutic proteins and host cell proteins (HCPs). The LYSE-BCT provides enhanced reduction and alkylation efficiency and all reagents are optimized to minimize artificial modifications such as oxidation or deamidation.

The LYSE-BCT and RESUSPEND-BCT reagent composition has been modified with reduction agent, detergent and pH being optimised to give improved performance. The iST-BCT CARTRIDGE is NOT suitable for sonication and is identical to the PHOENIX CARTRIDGE.

For even lower oxidation and deamidation rates and increased alkylation rate, reduce the temperature to 80°C and incubate for 10-20 minutes.

You can perform the LYSE and DIGEST steps in an appropriate plate or microcentrifuge tube until the addition of the STOP reagent. The total volume should then be transferred to the CARTRIDGE for the purify steps.

Protein content varies considerably across distinct biological input material. Different cell lines, strains, tissues, tissue regions, biological fluids and the sample storage conditions can affect the protein concentration. We recommend carrying out a protein concentration assay of the sample after the lysis step (see 1.7). A short overview of raw material amounts is given in the table below.
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We have a database with processing recommendations for >100 species and sample matrices. Please contact us for further details on your specific sample of interest.

Non-depleted human plasma and serum samples have a high protein concentration, commonly around 50 µg/µL. For processing of non-depleted plasma/serum for non-labeled the iST-BCT kit should be used, for labeled workflows use the iST-NHS kit. Mix 2 µL plasma/serum with 50 µL LYSE-BCT / LYSE-NHS buffer and continue with the regular iST-BCT / iST-NHS protocol.

Depending on the depletion process used, the resulting depleted plasma/serum sample may contain high concentrations of salts and/or have a large volume, making the samples incompatible for direct processing with PreOmics’ kits. We suggest performing protein precipitation (see 1.3) and using the resulting pellet as input material for the iST-BCT/iST-NHS protocol.

CSF samples have a wide reported concentration range, therefore PreOmics suggest performing protein quantification assay to determine the concentration prior to processing (see 1.7). For highly concentrated CSF samples, follow the recommendations for non-depleted plasma/serum (see 2.2) For diluted CSF samples (volume larger than 25 µL), perform protein precipitation (see 1.3) and continue with the regular PreOmics protocol. Both iST and iST-BCT are suitable for unlabeled workflows, iST-NHS should be used for labeled workflows.

Protein concentrations in urine samples vary substantially, we recommend either concentrating or precipitating the protein (see 1.3) before processing with the PreOmics kits. As a rule of thumb, 10-100mL human urine corresponds to ~100 µg protein, which needs to be concentrated down to 100 µL. Specifically for urine preparations, PreOmics provides an extra WASH0 buffer to effectively remove bilirubin contamination. Please inquire or refer to our website for an adapted protocol.

Protein concentrations in saliva samples vary widely, a rough estimate is about 10 µg/µL. Either collect ~10 µL saliva by spitting into a microcentrifuge tube or perform mouth swab and place the swap in 50-100 µL LYSE LYSE-NHS buffer to fully cover the swap. Keep all other buffer volumes as indicated in the regular protocols.

If extracellular matrix or transmembrane proteins are not of interest, you can use cell culture-grade trypsin to detach the adherent cells, wash them once with PBS and then store the cell pellet fraction as input for our regular protocols. Alternatively, if extracellular matrix or transmembrane are of interest and affected by partial trypsin digestion, add 50-100 µL of our LYSE/LYSE-NHS buffer directly to the cells and scrape them off. Collect the scraped material and heat to 95°C for 10 min before continuing with theregular iST/iST-NHS protocol.

Mammalian tissue samples are more difficult to lyse and require some kind of mechanical force disruption in the presence of our PreOmics’ LYSE buffers (). PreOmics’ BeatBox instrument efficiently and reproducibly homogenizes tissues and lyses cells from various sources. For more information, see https://www.preomics.com/beatbox.

FFPE samples can be easily processed by combining homogenization on the BeatBox with the iST technology. This workflow does not require a separate deparaffinization step with xylene. More details and a dedicated protocol can be found at https://www.preomics.com/applications/ffpe. FAQs related to this protocol are collected in section 11.

Plant tissues can be processed with our iST and iST-NHS kits but require cryogenic grinding or other means of mechanical force disruption initially. Specifically for plant preparations, PreOmics provides an extra WASH0 buffer to effectively remove secondary metabolite contamination, the protocol can be found here.

Enrichment of proteins via IP or co-IP strategies is compatible with our iST and iST-NHS technologies. Depending on the type of bead material used, the sample transfer to our cartridges happens either directly after the IP (transfer of proteins; magnetic beads) or after the digestion (transfer of peptides; agarose beads). For further information, please have a look at our two workflow recommendations available on the protocol tab here).

Model organisms can be entirely processed with our iST or iST-NHS kits. We recommend mechanical force disruption in the presence of our lysis buffers to effectively disrupt tissues/lyse cells. For processing of algae and diatoms, our WASH0 buffer removes secondary metabolites (provided upon request).

The lysis buffer in the iST-NHS kit, called LYSE-NHS, does not contain primary amines and therefore does not interfere with chemical labeling. The LYSE-NHS contains a distinct alkylation agent. Please consider the following as fixed modification in your database search:
Specific cysteine modification (C6H11NO), specificity [C], mass shift +113.084 Da

Chemical labeling experiments require very high peptide labeling efficiencies (>98%) for proper quantification. When struggling with lower labeling efficiencies, please see the following points:

There are two options for channel mixing:
A. Perform workflow until addition of STOP buffer, then mix channels accordingly. The pooled sample can be split equally on different CARTRIDGES (binding capacity of 100 µg per CARTRIDGE).
B. Perform workflow until end of vacuum concentrator step. Resuspend individual channels in LC-LOAD and then pool channels accordingly.
We advise to measure the peptide concentration of each channel for optimal channel pooling results.

Perform the labeling in an 1.5 mL microreaction tube and only to transfer the labeled peptides to our CARTRIDGES for the final peptide cleanup.

The iST-PSI kit contains two fixed well clean-up plates for the purification step on a positive pressure unit. The iST kit clean-up kit has an “array” plate format allowing the individual cartridges to be removed and processed in microcentrifuge tubes using adapters (not included but available for order  separately).

Additional buffers for the clean-up steps can be ordered separately if needed.
P.O.00109 iST-REG-PSI buffer add-on contains 2 x 100 mL of WASH1, WASH2 and ELUTE.

The iST-REG-PSI plates have been designed to work optimally when used with positive pressure, however they can also be processed by centrifugation.

The iST-BCT kit is used to prepare various biological fluid samples  for proteomic analysis, such as plasma, serum, and CSF. The ENRICH-iST kit allows deeper  insights into plasma and serum proteomics. It is founded on the bead-based enrichment of low-abundant proteins by breaking the dynamic range of plasma/serum proteome. ENRICH-iST combines a bead-based upstream enrichment step with iST-BCT sample preparation.

The ENRICH-iST kit has been specifically designed for  plasma and serum sample preparation.
The ENRICH-iST kit should be used when your target protein is low abundant, and its detection— if preparing the sample with iST-BCT—could be challenging.

ENRICH-iST 96x:
It has the flexibility to process batches from 1 to 96 samples. The DIGEST solution and buffers of the included iST-BCT are supplied in 2 mL tubes.

ENRICH-iST 96x HT:
It is used for high-throughput analysis to process 48 or 96 samples at once. The DIGEST solution is supplied in one vial, and all buffers are provided in 30 mL vials.

Yes, ENRICH-iST is compatible with different anticoagulants.  We recommend K2 EDTA or K3 EDTA as anticoagulants and blood harvest at RT; plasma should be prepared within 2 hours of blood collection by centrifugation at 2000 g for 10 min.

Plasma and serum samples derived from humans and  other mammalian species (i.e., mice, rats, pigs, and dogs) can be used.

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). 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.