We strongly advise to use the appropriate LYSE buffer provided with the PreOmics’ kits. If you want to use your own lysis buffer, this will probably adversely affect the efficiency of cell lysis and therefore protein identifications.
Please refer to the following table or contact us to check the compatibility of your lysis buffer with the iST system:

Your 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 of our 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.

For samples such as cells, yeast or tissue material, performing mechanical force disruption in LYSE or LYSE-NHS buffers will improve lysis efficiency. 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). We recommend the Bioruptor®Pico system (Diagenode), that allows direct placement of our iST and iST-NHS CARTRIDGES (please note iST-BCT and PHOENIX CARTRIDGES are not suitable).
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?"

The maximum volume is 250 µL.

Most classical assays are compatible with our PreOmics’ LYSE buffers. We recommend the BCA assay or the tryptophan quantification method. Some assays require dilution with distilled water to achieve the best results shown below:

Dilutions:
- BCA: none
- microBCA: 1:100 with PBS
- Bradford: 1:4
- Coomassie: 1:20
- Lowry: 1:4
- Tryptophan: none

For 20 µg protein starting material or less, add 10 µL LYSE and 10 µL DIGEST from the appropriate kit to your sample, keep all 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. We 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.

The digestion depends on your sample type and input material (see table below). Please note our recommendations to lower the enzyme amount for low input samples (<20 µg protein starting material, see "Which lysis temperature shall I use?".

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.

For simplicity reasons we recommend usage of buffer volumes indicated in the protocol. You can adjust the volume of LYSE and DIGEST to your starting amounts (e.g.: 20 µg protein starting material, 10 µl LYSE, 10 µl DIGEST, keep the other buffers as indicated in the protocol). For less than 20µg protein starting material, stay with 10µl LYSE and DIGEST, respectively. Accordingly, you may adjust the volumes for chemical labeling and quenching as recommended by the label manufacturer.

PreOmics have the PreON, a 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 with 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 PreOmics kits 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 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 "Are the PreOmics kits compatible with absolute quantification?"). Our peptide recovery is typically >80% for 1 - 100 µg protein starting material.

We perform strict QC measures for every buffer, kit and enzyme batch produced. Such 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, and more.
Upon request, PreOmics can provide 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 reaction: 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-NHS kits
◦ 4 reaction: 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 reaction: 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 reaction: 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. 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). TheLYSE-BCT provides enhanced reduction and alkylation efficiency and all reagents are optimised to minimise 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.

Protein content varies considerably across distinct biological input material (for example different cell lines, strains, tissues, tissue regions, biological fluids and the sample storage conditions the samples are subjected to (e.g. different storage temperatures, storage time, repeated freeze-thaw cycles,fresh frozen vs. FFPE, formaldehyde treatment). We advise to quantify protein concentration of your sample after the lysis step (see "Shall I adjust the buffer volumes depending on the protein starting amounts?"). A short overview of raw material amounts is given in the table below.
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 50µg/ µL. For processing of non-depleted plasma/serum for non-labeled the iST-BCT kit should be used, for labelled 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 "How do I perform protein precipitation?") 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 "Which protein quantification methods are compatible with the PreOmics’ kits?"). For highly concentrated CSF samples, follow the recommendations for non-depleted plasma/serum (see "How much raw material do I need for the PreOmics’ kits?") For diluted CSF samples (volume larger than 25 µL), perform protein precipitation (see "How do I perform protein precipitation?") 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 suggest either concentrating or precipitating the protein (see "How do I perform protein precipitation?") 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 substantially, we suggest either concentrating or precipitating the protein (see "How do I perform protein precipitation?") before processing with the PreOmics kits. As a rule of thumb, 10-100mL human saliva corresponds to ~100 µg protein, which needs to be concentrated down to 100 µL. Specifically for saliva preparations, PreOmics provides an extra WASH0 buffer to effectively remove bilirubin contamination. Please inquire or refer to our website for an adapted protocol.

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 using a rubber policeman. Collect the scraped material and heat to 95°C for 10 min before continuing with the regular 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 LYSE or LYSE-NHS buffers (see "Can I use mechanical force disruption in combination with the PreOmics’ kits?"). Addition of glass beads in combination with ultrasonication enhances the efficiency of tissue homogenization and cell lysis (Application notes can be found here).

Processing of FFPE samples requires de-paraffinization of the FFPE punches/slides. In addition, deparaffinized samples require a longer heat treatment for efficient de-crosslinking: increase the initial heating step from 10 to 60 min at 95°C. More details can be found in the FFPE protocol available here).

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 coIP 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:

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.

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.