Services and Fees

  1. Protein identification
  2. MW determination (MALDI or ESI-MS)
  3. Peptide sequencing
  4. PTM localization and characterization
  5. Sample preparation (Desalting, Digestion and Fractionation)
  6. Hourly rates (Open-access instruments and personnel)
  7. Relative protein quantification (label free, SILAC, iTRAQ,….)
  8. Absolute protein quantification – Assay devpt (SRM-MRM, AQUA, PSAQ)

No    Analysis type Sample type, applicability Output
1 Protein identification
11a NanoLC-MS/MS
short gradient (< 90min)
1D gel (typically bands from pull-down or CoIP) 1-100 Proteins
11b NanoLC-MS/MS
medium gradient (> 120min)
1D gel or LC fraction (typically bands from pull-down or CoIP) 1-500 Proteins
11c NanoLC-MS/MS
long gradient (> 240min)
Protein mixtures, cell extract, proteome subfraction, whole proteome 500-3000 Proteins
12 Multidimensional LC-MS/MS
Combination of item 52 with item 1
Protein mixtures, cell extract, proteome subfraction, whole proteome 500-5000+ Proteins
2 MW determination (MALDI or ESI-MS)
21a <10 samples MALDI Purified protein/simple mixtures List of masses
21b >10 samples MALDI Purified protein/simple mixtures List of masses
21c Large series MALDI
Open-Access
For trained users only
Purified protein/simple mixtures List of masses
21d Infusion ESI-MS/MS Infusion for small recombinant proteins (accurate mass) List of masses
21e Short gradient LC-ESI-MS/MS Short gradient for heterogeneous proteins (glycoproteins) List of masses
3 Peptide sequencing
31a Infusion ESI-MS/MS Purified peptide AA sequence
31b MALDI Purified peptide AA sequence
4 PTM localization and characterization
41a Phosphorylation Site Localization Bands of 1D and 2D gel, purified protein Report with localization site probability
41b Phosphorylation Site Localization Complex Simple and complex mixture sample (cells lysate,…) Report with localization site probability
5 Sample preparation (Desalting, Digestion and Fractionation)
51a Sample desalting – Tips
Sep-Pak cartridges
All samples Tips
Sep-Pack cartridges
51b Detergent removal on cartridges Ex: SDS detergent removal on Thermo cartridges Depends on cartridge size
52a InGel protein digestion Proteins separated on SDS-PAGE gel Included in items 11a-c
52b In solution protein digestion Proteins separated by LC or pI fractionation Included in items 11a-c
52c Filter Aided Sample Preparation (FASP) Complex protein mixtures in detergent and exotic buffers Pipeline coupled to items 53b-d and 11a-c
53a 1D-Gel with Coomassie/Silver
Staining
Protein mixtures 1st dimension separation Pipeline coupled to items 11a-c
53b Peptide Fractionation
SAX 6 fractions
Complex peptide mixtures 1st dimension separation Pipeline coupled to items 11a-c
53c Peptide Fractionation
SCX 12 to 24 fractions
Complex peptide mixtures 1st dimension separation Pipeline coupled to items 11a-c
53d Peptide Fractionation
OffGel (pI) 12 or 24 fractions
Complex peptide mixtures 1st dimension separation Pipeline coupled to items 11a-c
6 Hourly rates (Open-access instruments and personnel)
61a MALDI-TOF/TOF
Open Access hourly rate for trained personnel only
See 21c
61b Orbitrap XL
Open Access hourly rate for trained personnel only
Currently in test
61c Akta FPLC
Open Access rate for trained personnel only.
Contact Jean-Philippe Gaudry LMRP
jean.gaudry@epfl.ch
62 Personnel half-day bundle for assistance or extended work/per project Specialized databases, data post-treatment, pathway analysis, ontology enrichment, etc… Bioinformatics extended report
7 Relative protein quantification (label free, SILAC, iTRAQ,….)
71a Label Free – LC-MS/MS
Relative quantification
CELL CULTURE & TISSUE SAMPLES <> Complex mixture, Co-IP experiments Bioinformatics extended report
71b SILAC – LC-MS/MS
Labeling @ protein level (Duplex, Triplex)
Relative quantification
CELL CULTURE SAMPLES
Simple and complex mixtures (cell lysate,…), Co-IP experiments
Bioinformatics extended report
71c diMe labeling – LC-MS/MS
Labeling @ peptide level (Duplex, Triplex)
Relative quantification
CELL CULTURE & TISSUE SAMPLES Bioinformatics extended report
71d iTRAQ/TMT – LC-MS/MS
Labeling @ peptide level (Duplex, 4Plex, 8Plex)
Relative quantification
TISSUE SAMPLES
Simple and complex mixtures (cell lysate,…)
Bioinformatics extended report
72 PTM relative quantification
Label free, SILAC, diMe or iTRAQ (e.g. phosphorylation).
CELL CULTURE & TISSUE SAMPLES Bioinformatics extended report
8 Absolute protein quantification – Assay devpt (SRM-MRM, AQUA, PSAQ)
81a LC-SRM/MRM-MS
Absolute quantification
Stable Isotope Dilution
Peptides AQUA
Set of selected proteins Bioinformatics extended report
81b LC-SRM/MRM-MS
Absolute quantification
Stable Isotope Dilution
Proteins PSAQ
Set of selected proteins Bioinformatics extended report

Detailed explanation of analytical workflows and services

The words band and spot are used indifferently to designate a sample coming either from a 1D-Gel (band) or 2D-Gel (spot).

1 Protein Identification
11a
Protein identification by nanoLC-MS/MS from Gel (short gradient)
Starting from 1D-gel band cut from a gel lane, proteins are digested and the peptides extracted. The mixture is then separated on a nano-HPLC system on-line with an electrospray mass spectrometer (LTQ OrbitrapXL, Elite or QExactive), which isolates and fragments as many peptides as possible during an 80-min gradient. Collections of MS/MS spectra are used for database search for protein identification. This workflow is used for analyzing simple to medium complexity mixtures (a few hundred proteins) as well as for searching for modified peptides (PTM’s). This workflow is the main workflow for low complexity samples (CoIP experiments) or samples isolated from species with incomplete genome sequences when sequence coverage is needed or PTM detection is necessary (additional fee for manual work might apply here: see item 62).
11b-c
Protein identification by nanoLC-MS/MS from Gel or LC fraction (longer gradient)
Same as 11a starting from an LC fraction or 1D-gel band from a complex sample (ex: total protein extract) but with gradients longer than 80 min. This workflow is also applied to Protein identification by nanoLC-MS/MS from LC fraction (medium complexity). Proteins are digested and the peptides extracted. The mixture is then separated on a nano-HPLC system on-line with an electrospray mass spectrometer (LTQ OrbitrapXL, Elite or QExactive), which isolates and fragments as many peptides as possible during a 120- or 240-min gradient. Collections of MS/MS spectra are used for database search for protein identification. This workflow is used for analyzing medium to high complexity mixtures (between a few hundred to several thousand proteins) as well as for searching for modified peptides (PTM’s). This workflow is the main workflow for complex samples isolated from species with incomplete genome sequences when sequence coverage is needed and PTM detection is necessary (additional fee might apply here: see hourly charge for special work in item 62).
12
Multidimensional analysis of complex fractions (Proteome in-depth characterization)
The protein pool is reduced, alkylated and digested. The peptides are then separated in a first dimension (SAX, pI, SCX, etc…) in several fractions as described in item 5. Analysis is performed by LC-MS/MS on every fraction as in workflow 11a or 11b. The resulting collections of spectra are pooled for every sample before database search. Lists of identified proteins for each sample with their scores are subjected to statistical validation and aligned for comparison.

Note 1: a negative control is mandatory for background subtraction for the analysis of protein complexes.

Note 2: contact us well in advance if you plan to undertake such studies.

2 MW determination (MALDI or ESI-MS)
21a
Protein and peptide(s) MW determination by MALDI-MS
This simple analytical workflow is used to determine the accurate molecular weight and the heterogeneity of a peptide or a protein after an optional desalting step by direct MALDI-TOF MS analysis. It usually works well on recombinant proteins with masses up to 60’000 Da (depends on the heterogeneity and the amount). Requires however relatively concentrated samples with low detergent concentration (pmol range). The analysis includes up to 2 attempts with different matrices.
21b
Same as 21a
A special rate with has been introduced for groups submitting more than 10 similar samples the same day. Care should be taken to submit samples with low salt content as desalting steps performed by PCF personnel will be charged extra (see item 51a).
21c
Protein and peptide(s) MW determination by MALDI-MS (Open-Access option)
The facility offers training in MALDI sample preparation for users who would like to submit their sample directly prepared on plate and perform their own analyses. The training is worth considering for groups submitting large sample series regularly as special rate apply for all samples submitted that way. Only the hourly rate for instrument use is applied (100.- CHF on the ABI4800). An extra charge of 2.- CHF per sample for consumables is applied if the samples are desalted and prepared in the PCF lab.
21d
Protein and peptide(s) MW determination by Infusion ESI-MS
This analytical workflow is used to determine the accurate molecular weight of a peptide or a small protein after a desalting step by direct infusion in an ESI-MS instrument for masses up to 45’000 Da (Triversa). It usually provides better mass accuracy than workflow 21a on small recombinant proteins with masses ranging from 10 up to 45 kDa but is more sensitive to the heterogeneity and the amount. The analysis requires relatively concentrated and detergent-free samples (pmol range). Additional pretreatment will be charged for sample containing detergents (see item 51b).
21e
Protein and peptide(s) MW determination by short gradient LC-MS
This analytical workflow is used to determine the accurate MW of simple mixtures of small proteins or for larger proteins that need extensive desalting (glycoproteins, immunoglobins, etc…). The analysis requires relatively concentrated and detergent-free samples (pmol range). Additional pretreatment will be charged for sample containing detergents (see item 51b).
3 Peptide sequencing
31a
Peptide(s) sequencing by Infusion-ESI-MS/MS
Purified peptides are first desalted on StageTip C18 column. The peptide(s) is analyzed on a Triversa nanoES source in infusion mode on an electrospray mass spectrometer (LTQ OrbitrapXL) and extensively fragmented. The sequence is then deduced from the different tandem MS spectra by DeNovo sequencing. This workflow is perfectly adapted for final sequence assessment of synthetic peptides.
31b
Peptide(s) sequencing by MALDI-MS/MS
Purified peptides are first desalted on StageTip C18 column. The peptide(s) is analyzed on the ABI 4800 MALDI-TOF/TOF and extensively fragmented. The sequence is then deduced from the different tandem MS spectra by DeNovo sequencing.
4 PTM localization and characterization
41a
Phosphorylation site analysis (simple)
Gel-separated or liquid phase samples are digested (if necessary with several proteases) and phosphopeptides are enriched by an affinity step on a titanium dioxide microcolumn (other enrichment methods such as IMAC are under evaluation). A fraction of the eluate from the TiO2 column is first analyzed either by MALDI-TOF/TOF or infusion ESI-MS/MS to assess the enrichment, then by nano-LC-MS/MS (HCD/CID)) to identify and localize the modification site(s) on a maximum of phosphorylated peptides. A fraction of the non-enriched peptide mixture is always processed in parallel to maximize the protein identification rate (2 LC-MS/MS injections). Digestion with multiple enzymes might be necessary to narrow down the position of some phosphorylation sites. Extended gradient length might be necessary to maximize the output in the case of complex samples.

Note: an appropriate negative control (non-phosphorylated) should be supplied whenever possible. The analysis of the negative control will however be charged as two samples have to be analyzed.

41b
Phosphorylation site analysis (complex)
The samples are treated as in 11b. Phosphopeptides are first enriched by a specific SCX step which fractionates all the peptides. SCX fractions are then further enriched by one or 2 affinity steps in microcolumns (TiO2). The eluates of these affinity columns are then analyzed as in item 1 using the appropriate instruments and gradient length. Hundreds to thousands phosphorylation sites can be characterized that way.

Note 1: the number of phosphopeptides and phosphorylation sites assigned with confidence is largely dependent on the amount of starting material.

Note 2: consultation with PCF-PTP personnel mandatory prior to sample submission since this pipeline needs to be adjusted to the objective of the request. Contact us well in advance if you plan to undertake such studies. The facility can help you with the experimental design.

5 Sample preparation
51a
Sample desalting (tips and SepPak)
Samples containing low salt buffers usually work better and lead to less issues at the MS analysis stage. Whenever necessary, a sample desalting step before MS analysis will be performed by off-line liquid chromatography (reversed-phase or other). This sample treatment step will be charged if performed by PCF-PTP personnel. We therefore encourage our users to perform this step by themselves prior to sample submission. Material, consumables and advices are available at the facility if not available in your laboratory. For larger amounts, SepPak cartridges taken from PCF-PTP stock will be charged as consumable. Unless otherwise stated, desalting steps will always be charged extra for all services.
51b
Detergent removal on cartridges
Samples containing detergents like SDS often lead to issues at the LC-MS analysis stage. It is recommended to minimize the amount of detergent at the sample preparation stage whenever possible. Some detergents like SDS can however be efficiently removed by treating the mixture with special cartridges. This sample treatment step will be charged if performed by PCF-PTP personnel. We therefore encourage our users to inform PCF-PTP personnel about the presence of detergent in their samples during submission. Material, consumables and advices are available at the facility if not available in your laboratory. Detergent removal on cartridges will always be charged extra for all service.
52a
InGel protein digestion
This sample preparation step comprises gel destaining, reduction alkylation, digestion, peptide recovery and concentration. The setup fee covers the preparation steps and the per-sample fee, the consumables. These charges are detailed here for information. They are included in most of the cases in item 1. Digestion with an alternative enzyme other than trypsin performed separately on the same sample is considered as a new sample.
52b
In solution protein digestion
This sample preparation step comprises reduction alkylation, digestion, peptide recovery and concentration. The setup fee covers the preparation steps and the per-sample fee, the consumables. These charges are detailed here for information. They are included in most of the cases in item 1. Digestion with an alternative enzyme other than trypsin performed separately on the same sample is considered as a new sample.
52c
Filter-Aided Sample Preparation (FASP)
This efficient sample preparation is recommended for samples prepared in exotic buffers containing detergents as it is often the case for membrane proteins or total cell extracts. This sample preparation consists mainly in replacing the detergent containing buffer by urea that is performed on filters with 10 or 30 kDa MW cutoff. Digestion is then performed on the filter using the adequate enzyme(s). Up to 500 µg of sample can be processed with the smallest version of the filter. For larger amounts up to 3 mg of proteins, a larger filter form factor is available. This procedure is often coupled to item 53b and 53c.
53a
1-D SDS-PAGE Gel separation
Protein mixture containing detergents can be treated by running 1D-Gel separations on standard minigels on short distance to clean up the sample. This sample treatment step will be charged if performed by PCF-PTP personnel. Gel bands are then treated as in item 11a-c. We therefore encourage our users to perform the gel separation themselves prior to sample submission. Please do not forget to use MS-compatible staining kits. Material, consumables and advices are available at the facility if not available in your laboratory.
53b
Peptide fractionation – SAX 6 fractions
Complex peptide mixtures are fractionated off-line on Strong Anion eXchange phase in special StageTips. The resulting fractions are then often analyzed as in item 11b or 11c. Bundle prices apply to the LC-MS/MS analysis.
53c
Peptide fractionation – SCX 12 to 24 fractions
Complex peptide mixtures are fractionated off-line on a Strong Cation eXchange column. The resulting fractions are then desalted as in item 51a and analyzed as in item 11a-c. The number of fractions depends on the amount of starting material and analysis depth to be achieved. A special optimized gradient is used prior to phosphopeptide enrichment as detailed in item 41b. Bundle prices apply to the LC-MS/MS analysis.
53d
Peptide fractionation – pI separation by OffGel 12 or 24 fractions
Complex peptide mixtures are fractionated off-line according to their isoelectric point on an OffGel system. The number of fractions depends on the amount of starting material and analysis depth to be achieved. The resulting fractions are then desalted as in item 51a and analyzed as in item 11a-c. Bundle prices apply to the LC-MS/MS analysis.
6 Hourly rates (Instruments and personnel)
61a-c
Instrument Open-Access rates
See item 21c for 61a. Open-Access to the Orbitrap XL instrument is under evaluation. Open-access on these instruments is only available to personnel that have successfully completed the training.
62
Personnel hourly rate for assistance and extended work
Manual DeNovo sequencing, post-translational modification localization, special database creation and pathway analysis can quickly become time consuming especially when only low amounts of sample are available for analysis. This service will be charged in addition to the fee-for-services 11a-c, 12, 31a-b, 41a and 41b if the processing time exceeds 1 hour.
7 Relative protein quantification (label free, SILAC, iTRAQ,….)
71a
Label Free – LC-MS/MS quantitative workflow
This quantitative workflow is purely bioinformatic and uses several algorithms to derive the relative quantities. Any protein identification analyses performed according to item 1 with enough replicates can be analyzed that way. Best statistical precision can only be achieved if enough replicates can be processed. Pricing is dependent on the time spent to process and interpret data (item 62).

Note: preliminary consultation with PCF-PTP personnel is advisable at the experiment planning stage if you want to achieve good precision. Contact us well in advance if you plan to undertake such studies.

71b
SILAC – LC-MS/MS quantitative workflow – Labeling @ protein level
SILAC is an accurate quantitative proteomics technique based on stable isotope labeling with amino acids in cell culture. It is mainly applicable to mammalian cultured cells but offers great advantages for experiments in which complex purification steps are required to isolate a fraction of interest (Co-IP), as well as for general quantitative profiling. Up to three conditions can be encoded this way. The PCF-PTP offers expert support during the setup of SILAC labeling experiments with guidance through preliminary experiments and appropriate bioinformatics tools to establish cell culture conditions. The PCF-PTP supports the full analytical pipeline (including data analysis and advice in preliminary interpretation). Carrying out cell culture and the first steps in sample preparation remain the customer’s responsibility. Best statistical precision can only be achieved if enough replicates can be processed. Pricing is dependent on the degree of fractionation needed to achieve adequate analytical depth (number of fractions (item 5) and gradient length (item 1)).

Note 1: the PCF-PTP maintains a stock of stable isotopes labeled amino acids that are available at very attractive prices.

Note 2: preliminary consultation with PCF-PTP personnel mandatory prior to sample submission. Contact us well in advance if you plan to undertake such studies.

71c
diMe labeling – LC-MS/MS quantitative workflow – Labeling @ peptide level
diMe labeling is a post-digestion stable isotope labeling technique which consists in adding methyl groups to primary amines on peptides (reductive amination). DiMe labeling is an accurate quantitative proteomics technique applicable to any kind of protein mixture (protein extracts from cells and tissues). Up to three conditions can be encoded this way. The advantage of this technique is in the low price of the labeling reagent which makes it an advantageous option when large amount of sample (mg) needs to be labeled. This workflow is compatible with most peptide fractionation methods (item 5) and phosphorylation enrichment methods (item 4). Best statistical precision can only be achieved if enough replicates can be processed. Pricing is dependent on the degree of fractionation needed to achieve adequate analytical depth (number of fractions (item 5) and gradient length (item 1)).

Note: preliminary consultation with PCF-PTP personnel mandatory prior to sample submission. Contact us well in advance if you plan to undertake such studies.

71d
iTRAQ-TMT labeling – LC-MS/MS quantitative workflow – Labeling @ peptide level
iTRAQ or TMT labeling is a post-digestion stable isotope labeling technique which consists in adding special labels with isobaric mass to primary amines on peptides. Quantification is performed at the MS2 level using diagnostic fragment ions of the labels. Up to 10 conditions can be simultaneously analyzed per one single run. iTRAQ-TMT is mainly used when many conditions need to be tested simultaneously and is applicable to a wide variety of samples that are not easily labeled at the protein level (tissues and biofluids). This workflow is compatible with most peptide fractionation methods (item 5) and phosphorylation enrichment methods (item 4).

Note 1: this workflow is still under development.

Note 2: preliminary consultation with PCF-PTP personnel mandatory prior to sample submission. Contact us well in advance if you plan to undertake such studies.

72
PTM relative quantification
This workflow is actually a combination of item 4 with item 5 and item 7 (71a-d) that is adapted to the amount of starting material available and the analysis depth requested. Item 62 often needs to be applied as data interpretation is not always straightforward.

Note 1: this workflow is still experimental and needs active involvement of the group requesting the analysis.

Note 2: preliminary consultation with PCF-PTP personnel mandatory prior to sample submission. Contact us well in advance if you plan to undertake such studies.

8 Absolute protein quantification – Assay devpt (SRM-MRM, AQUA, PSAQ)
81a
Absolute (accurate) quantification by LC-SRM/MRM-MS using AQUA peptides
This workflow consists in developing an assay specifically tailored for the accurate quantification of a set of proteins selected for their interest (biomarkers, proteins involved in a pathway or a metabolic path, etc…). Proteins are quantified at the peptide level using stable isotope dilution techniques on triple quadrupole instruments (TSQ Vantage). The facility is working in collaboration with several providers that can deliver the adequate synthetic peptides – accurately quantified or not – that are used either to set-up the assay or to perform the quantification. The assay development phase comprises the selection of a set of adequate proteotypic peptides, the procurement of the right peptide standards, the optimization of SRM measurement and the evaluation of the robustness and accuracy of the assay. Each consumable is billed at real cost recovery. LC-SRM-MS analyses are charged on a per hour basis. Please contact us if you want more information.

Note 1: this workflow involves several steps and is still experimental. Assay development needs active involvement of the group requesting the analysis.

Note 2: assay development for accurate quantification of a defined set of proteins is only worth the investment if several experimental conditions or biological replicates need to be measured.

Note 3: preliminary consultation with PCF-PTP personnel mandatory prior to sample submission. Contact us well in advance if you plan to undertake such studies.

81b
Absolute (accurate) quantification by LC-SRM/MRM-MS using stable isotope labeled proteins (PSAQ approach)
This workflow is actually very similar to workflow 81a but makes use of stable isotope labeled proteins instead of peptides for better quantification accuracy. The PCF-PTP has developed a methodology in collaboration with the group of Prof. Bart Deplancke (EPFL) that combines the production of heavy labeled proteins using in vitro translation and in situ quantification of the freshly produced protein standard using a quantification tag. Other sources of accurately quantified heavy labeled proteins are under evaluation at this stage. Please contact us if you want more information.