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iST technology for sample preparation

Is my own lysis buffer compatible for use with the PreOmics’ kits?
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:
Own lysis buffer
PreOmics compatible?
Max. volume lysis buffer
Considerations
PBS
Yes
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
RIPA (max. 0.1% SDS, 1%SDC, 1% Triton x-100)
Yes
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
Urea (max. 2M; no thiourea)
Yes
25 µL
dilute with equal volume of appropriate 2X concentrated LYSE reagent
Thiourea
No
-
perform protein precipitation
High salt (e.g. >0.5M NaCl)
No
-
perform protein precipitation
What is the maximum starting volume of my sample to be processed with the PreOmics kits?
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.
How do I perform protein precipitation?
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
Can I add protease inhibitors to my sample?
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.
Can I use mechanical force disruption in combination with the PreOmics’ kits?
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.
What are the minimum and maximum protein amounts for the PreOmics’ kits?
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?"
What is the maximum volume I can load on the PreOmics’ CARTRIDGES?
The maximum volume is 250 µL.
Which protein quantification methods are compatible with the kit?
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
Shall I adjust the buffer volumes depending on the protein starting amounts?
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.
Which lysis temperature shall I use?
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).
Can I use other enzymes for the protein digestion?
The PreOmics’ kits come with a lyophilized enzyme mix consisting of LysC and trypsin.
How long shall I digest my samples?
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?".
Sample type
Digestion time
Precipitation proteins
1-3 hrs
Cell lines
1-3 hrs
Biological fluids
1-3 hrs
Tissues (mammalian, plants)
3 hrs
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.
Are the PreOmics’ workflows compatible with enrichment of phosphorylation sites?
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.
Should I adjust the volumes of the buffers with protein starting amounts?
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.
How can I automate my sample processing efforts?
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.
Are the PreOmics kits compatible with absolute quantification?
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.
How do I assess the peptide recovery rate after the whole sample processing workflow?
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.
Which kind of quality control does PreOmics provide for the kits?
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.
What are the differences between the 4x, 8x, 12x, 96x and 192x PreOmics’ kits?
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
Are the PreOmics kits compatible with peptide fractionation?
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.
When should I use the iST-BCT kit?
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.
What is the difference between iST and iST-BCT kits?
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.
Protocol tip to further minimize artificial modifications when using iST-BCT
For even lower oxidation and deamidation rates and increased alkylation rate, reduce the temperature to 80°C and incubate for 10-20 minutes.

Sample type recommendations

How much raw material do I need for the PreOmics' kits?
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
Material
Starting amount
Protein amount
Mammalian Cell Line (e.g. HeLa)
6E5 cells
100 µg
Yeast (S.cerevisae)
OD600 = 0.6
100 µg
Bacteria (E.coli)
OD600 = 0.5
100 µg
Immunoprecipitation
1 mL slurry
10 - 400 µg
Blood / Serum / Plasma (H.sapiens)
2 µL
100 µg
Mammalian Tissue
1 mm3
100 µg
Plant Tissue (A.thaliana): shoot/root wet weight
50 mg / 100 mg
100 mg / 100 mg
How do I process non-depleted plasma/serum samples?
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.
How do I process depleted plasma/serum samples?
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.
How do I process CSF samples?
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.
How do I process urine samples?
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.
How do I process saliva samples?
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.
How do I process adherent cell culture samples?
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.
How do I process mammalian tissue samples?
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).
How do I process FFPE samples?
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).
How do I process plant tissue samples?
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).
How do I process immunoprecipitation samples?
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).
How do I process bacteria/yeast/algae/diatoms?
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).

Chemical labeling (iTRAQTM, TMTTM)

What is the difference between the iST and the iST-NHS kits?
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
How can I improve the labeling efficiency when performing chemical labeling in combination with the iST-NHS kits?
Chemical labeling experiments require very high peptide labeling efficiencies (>98%) for proper quantification. When struggling with lower labeling efficiencies, please see the following points:
  • Make sure that the sample input material (e.g. cell pellet) is not contaminated with residual buffersor cell culture media containing primary amines that interfere with chemical labeling.
  • Use fresh labeling reagents to achieve the highest labeling efficiency. Resuspended labeling agent,which is not used throughout the experiment, should not be stored longer than two weeks at -20°C. Resuspended labeling reagent will hydrolyze over time leading to lower labeling efficiency.
  • Use TMT at a label to peptide ratio of 4:1, i.e. 400 µg of TMT label per 100 µg of peptides. Higher ratios will slightly increase the labeling efficiency but commonly reduce peptide identification rates
  • Use an acetonitrile (ACN) concentration of at least 30% during the labeling reaction, i.e. 50 µL LYSE+ 50 µL DIGEST + 42 µL of labeling reagent in dry ACN. Lower amounts of ACN will quickly hydrolyze the labeling agent. If you have resuspended the labeling reagent in a smaller ACN volume, add some dry ACN to the solution in order to achieve a final concentration of 30% ACN.
    Take the volume of the cell pellet and residual buffer/cell culture media into account for the calculation of the final ACN concentration.
Shall I perform the chemical labeling step on the cartridge or in the tube?
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.
When shall I mix channels after the chemical labeling?
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.

Peptide Cleanup

Which sample types require additional wash steps?
Most peptide samples can be cleaned up with the washing buffers presented 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 stronger sources 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”.
What are the differences between the PreOmics washing buffers?
The Phoenix peptide cleanup kit is able to remove detergents, sugars, lipids, salts and various polymers. For more details see our Phoenix Application Note.
Washing buffer
Organic
Acidic
Basic
Volatile
Sample types
WASH0
Yes
Yes
Yes
urine, plants
WASHX
Yes
Yes
Yes
lipids, polymers, detergents
WASH1
Yes
Yes
Yes
hydrophobic contaminations
WASH2
Yes
Yes
hydrophilic contaminations
How do I elute my samples from the 96-well CARTRIDGE adapter plates?
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.
How long do I need to concentrate my samples in the vacuum concentrator?
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.
Can I concentrate PreOmics’ eluates together with samples eluted from C18 columns in the same 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.
Which peptide quantification methods can I use after processing my samples?
Peptide quantification should be done in our LC-LOAD buffer and not in the ELUTE buffer. We recommend quantitative colorimetric peptide assays.
How can I resuspend dried peptides in the 96-well plate?
After the vacuum concentrator step, you can add our LC-LOAD buffer to each well and shake in a horizontal plate shaker (500 rpm, 5 min).
How shall I store resuspended peptide samples after processing them?
Storage of peptides should not exceed two weeks at -20°C. For long-term storage, keep them at -80°C.

Phoenix kit

Are the CARTRIDGES from the iST, iST-NHS, iST-BCT and PHOENIX kits the same?
CARTRIDGES in the iST and iST-NHS kits are the same. CARTRIDGES in the iST-BCT and PHOENIX kits are of different nature and have a slightly lower affinity for hydrophobic species.
How do I load my peptide samples on the PHOENIX CARTRIDGES
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

PreON automation platform

What are the pre-installation requirements?
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.) x62 cm (24.4 in.) x 86 cm (34.0 in.) with a weight of 71.5 kg (157.6 lbs).
Which sample types can be processed on the PreON?
The PreON can process protein pellets, intact cells, body fluids and tissue lysates.
How many samples can I process on the PreON per day?
The sample throughput is 12 samples per run with the option to execute multiple runs per day. For iST workflows, up to three runs per day with a total of 36 samples are feasible. For iST-NHS workflows,up to two runs per day with a total of 24 samples are feasible.
Can the PreON prepare both label-free and chemical labeling samples?
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.
Can I run my own methods on the PreON?
The PreON works seamlessly with our ready-to-go iST, iST-BCT and iST-NHS kits. Other methods or commercial products cannot be operated with the PreON.
Which kind of maintenance does the PreON require?
The PreON requires minimal maintenance such as cleaning the surfaces and emptying the solid and liquid waste containers. Once every six months we recommend to perform a tightness test for the pipetting unit (all required utensils provided).
How is the PreON serviced?
Our global and trusted partner PEAK-Service is the leading global provider for technical services for medical, analytical and industrial equipment. Contact us to inquire about further servicing options and pricing.

Accessories for PreOmics technologies

What is the Metal Heating Shaker Adapter?
The Metal Heating Shaker Adapter guarantees optimal heat transfer for our CARTRIDGES compared to planar heating systems. It is compatible with any heating shaker in the SPSS format and many liquid handling platforms. It is also directly compatible with our 96-well CARTRIDGE adapter plates.
When purchasing the PreOmics’ 96 reaction kits, do I need to order the 96-well adapter plate too?
All our kits in the 96 reaction format already contain the 96-well adapter plate required for convenient handling of larger sample numbers.

Ordering & Shipping

How can I purchase products from PreOmics?
Customers from North America please order via PreOmics Inc. (USA), customers from the rest of the world please order via PreOmics GmbH (Germany)

For the convenience of our customers, we offer several ways of purchasing our products:

- Order by email: order@preomics.com

- Order by phone: +49-89-2314163-0

- Order by fax: +49-89-2314163-99
Does PreOmics ship worldwide?
Yes, we ship our products worldwide from Germany. For our US customers, we drop-ship directly from our warehouse in New Jersey.
Is it possible to order individual items from your kits?
We provide complete solutions to ensure best results for your LC-MS/MS analyses. Adaptions to our lysis buffers might be necessary though for specific experimental questions. Upon request, we provide the appropriate 2x concentrated LYSE buffer and the WASH0 for use with plant, algae and diatom samples.

Any more questions?

Don’t hesitate, contact us!