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Services and Fee Schedule

Upstate Investigators

Before preparing the samples, please read the sample preparation guidelines and requirements for an application of interest or contact the facility.

Consultations are free.

Service Type Unit Price, $
Protein digestion* 15+
Microscale cleanup 15
MW determination 50
Protein identification short gradient 75
medium gradient 125
long gradient 200
Phosphopeptide enrichment 75
Protein profiling TMT,
iTRAQ, or SILAC analysis
1D 200
2D** 2400
Method development 60/h
Data analysis 60/h
Targeted analysis (MRM or SRM) 20
LC-HRMS on Lumos 50

*for each batch, add $45 for in-gel or $40 for in-solution digestion set-up fee
**cost is $200 per fraction.  Cost shows reflects a 12-fraction (typical) analysis

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Protein Digestion

Proteolytic digestion is prerequisite for protein identification and quantification by LC/MS/MS analysis. Proteins are digested in solution or in gel band/spot.

The most commonly used enzyme for protein digestion is trypsin. Digestion can be performed by the facility for a charge of $15 per sample and a per-batch fee of $40 or $45. Typically, this will be followed by a microscale sample cleanup and a short or medium-length LC-MS/MS analysis.

The following services are provided for trypsin digestion:

Service Fees Sample Requirements
In-gel tryptic digestion  $45/set-up + $15/sample
  • Gel thickness: 1mm
  • Total protein in a sample: a protein band visible by Coomassie or silver staining
In solution tryptic digestion $40/set-up + $15/sample
  • Total protein in a sample: ≥ 200 femtomole/protein
  • Total protein content in a sample: 10 to 250 µg
  • Samples should not contain detergents

Molecular Mass Determination

Dissolved analyte (e.g. protein, peptide, or small molecule) is infused into the mass spectrometer, the mass to charge ratios (m/z) are measured, and the molecular masses are determined. Volatile solvents facilitating positive (proteins, peptides, small molecules) or negative (small molecules) ionization of analytes are used.  Salts and detergents are not compatible with this analysis and therefore should be removed.

The following services are provided:

Service Fees Sample Requirements
MS analysis - direct sample infusion; determination of the molecular mass of an analyte (small molecule, protein, or peptide).
  • Samples can be submitted either as soluble lyophilized powder or in solution with adequate volatile buffer
  • Analyte concentration > 5 µM
  • Protein/peptide sample buffer: 0.1% formic acid, 5% (or higher) acetonitrile
  • Samples should not contain detergents, non-volatile salts, etc.

Protein Identification

Proteins are most commonly identified by so-called bottom up proteomics, referred to as “shotgun proteomics”.  Ultimately, information from peptide fragments are used to assemble peptide sequences and finally intact proteins – hence the name “bottom up”.   As peptides behave more ideally during chromatographic separation and mass analysis than do proteins, it is advantageous to enzymatically digest samples prior to analysis. 

Samples are first digested using a sequence-specific protease, usually trypsin.  All non-peptide material has the potential to interfere with their detection, and salts and detergents are notoriously problematic. Fortunately, a variety of techniques can successfully remove most of these contaminants, and all digests should be followed by a clean-up step.  The resulting mixture of peptides must be simplified before introduction into the mass spectrometer, and this is achieved by online nanoscale liquid chromatography (nLC).  The mass spectrometer detects peptide signals in real time as they emerge from the nLC column.  However, in order to determine the amino acid sequence, the peptides must be isolated in the gas phase, fragmented, and the masses of these fragments measured in a tandem or MS2 spectrum.

It is these spectra which contain information about the peptide sequence. Several algorithms have been developed to automatically compare an experimentally-obtained spectrum to theoretical spectra from a protein database.  The matches are scored and filtered to achieve an acceptable false discovery rate.  The identified peptides are parsimoniously mapped back to their parent proteins to produce the set of putatively discovered proteins in the sample.

The following services are provided for protein identification:

Services Fees Sample Requirements
LC/MS/MS analysis of digested protein samples
  • Total protein in a sample: ≥ 200 femtomole/protein
  • Total protein concentration in asample: ≥ 10 µg
- Short Run (45 min gradient) $75/run
- Medium run (1.5 hr. gradient) $125/run
- Long run (2-3 hr. gradient) $200/run

Phosphopeptide enrichment

Protein phosphorylation plays a key role in cellular signaling and response to stimuli.  Large-scale phosphoprotein studies require enrichment of the low-abundance phosphopeptides prior to LC-MS/MS analysis.  A typical input for such an enrichment is 1 mg of protein, resulting in ~1-10 µg of phosphopeptides.

Service Fees Sample Requirements
Phosphopeptide enrichment $75 • Total protein required: ~1 mg

Quantitative proteomics - TMT Analysis

Protein profiling determines the relative abundance of proteins in complex protein mixtures. Using TMT analysis, the relative abundance of proteins can be determined in up to 10 protein extracts at a time (per analysis). This is achieved by labeling of complex protein mixtures using 10 mass spectrally resolvable, isobaric labels. TMT labeling is performed using a commercial kit (Thermo Scientific) according to the manufacturer’s protocol. Equal amounts (20-100 µg) of total protein from each sample are separately digested with trypsin and labeled with one of TMT reagents.  The labeled samples are pooled and analyzed by LC/MS/MS for protein identification and quantitation:  identical peptides with different TMT tags are resolved in the same chromatographic peak, the masses of eluted peptides are determined, and the peptides are fragmented.  The spectra of the fragmented peptides contain both sequence-informative ions and reporter ions which are used for quantification.

As researchers seek a “deep dive” into their biological system, TMT experiments often warrant a two-dimensional separation of the peptides.  The first, offline, separation can be scaled based on the researcher’s goals.  This can range in complexity from in-house assembled chromatographic media, to the use of an HPLC system with fraction collection.  The resulting fractions, usually numbering in the range of 6-20, are then individually analyzed by LC-MS for the greatest achievable coverage.

Service Fees Sample Requirements
TMT labeling and mixing Market price 20-100 µg of protein per sample, digested
Fractionation of a labeled, complex sample. Cleanup, QC, and LC-MS/MS analysis $200/fraction Mixed TMT peptides

Quantitative proteomics - SILAC Analysis

Stable isotope labeling by amino acids in cell culture (SILAC) is used for metabolic labeling of proteins for comparative, MS-based quantitative proteomics.  Proteins are labeled through metabolic incorporation of isotopically “light” or “heavy” forms of a given amino acid (usually lysine and arginine).  Two cell populations are grown in media with different labels, and labeled protein samples are isolated. Equal amounts of protein samples are pooled and digested by trypsin. Pooled, labeled, tryptic peptides are analyzed by LC/MS/MS for sequence identification and quantification:  identical “light” and “heavy” peptides derived from different samples are resolved in the same chromatographic peak, the masses and the intensities of eluted peptides are determined for quantification, and the fragment ions are used for sequence identification. Protein identification takes place as described above, but the software also determines a relative ratio of protein abundances in the sample.

As with chemical labeling (see TMT analysis, above), such experiments are often analyzed using two-dimensional chromatography.

The following service is provided for SILAC analysis:

Service Fees Sample Requirements
LC/MS/MS differential protein expression analysis of SILAC labeled/digested samples $200/fraction • SILAC labeling should only be performed using a commercial kit according to the manufacturer's protocol. Equal total amounts of labeled proteins (> 5 µg) are mixed and digested with trypsin, analyzed by LC-MS/MS

Small molecule quantification: Targeted analysis

Samples (cell lysate, biofluids) depleted of macromolecules are submitted to the facility. Small molecules of interest are quantified using LC/MS/MS approach. Analytes are separated by UPLC and monitored by tandem mass spectrometry using multiple reaction monitoring (MRM, also called SRM).  The instrument used, a triple quadrupole, has two mass filters.  Programmed to act synchronously, the first mass filter selectively admits the molecule of interest, while the second filter selectively targets known fragment masses.  This process is both sensitive and selective.  The instrument can easily be programmed to detect tens of analytes in a single analysis, and with some work, hundreds.

Two strategies exist to make the assay quantitative: internal and external calibration.  When using the former, a known amount of a heavy-isotope version of the target analyte is spiked into all samples.  The ratio of signals from the two isotopologues produces extremely accurate quantitative results.  When the acquisition of an isotopically-labeled standard is impractical, external calibration can be achieved by producing a calibration curve.  Here it is critical to provide a blank matrix, matching that of the samples.  This matrix is spiked with various amounts of a pure standard of the analyte.  The spiked calibrants are run at the beginning and end of a batch of samples, and quantification is achieved by relating the amount of signal in the samples to the regression line of the calibration curve.

These projects typically require significant up-front investment in method development (5-10h for a low number of analytes), but have relatively inexpensive rates for the analysis of large sample batches.

The following services are provided for small molecule quantification:

Service Fees Sample Requirements
Method development $60/hour 1-10 mg of pure standard and several aliquots of blank matrix
LC-MRM analysis $20/run Clear extracts/solutions free of macro molecules in volatile solvents

Protein quantification: Targeted analysis

As with small molecule quantification, proteins can be quantified in a targeted fashion.  Protein samples are digested to peptides, and a small number of peptides are chosen to represent the protein from which they originate.  Samples are spiked with isotopically-labeled peptides identical in sequence to those of interest.

Pricing is identical to small molecule quantification, but users should anticipate the extra costs of protein digestion, acquisition of labeled peptides, and a more involved method development.

LC with high resolution MS

For use with untargeted metabolomics, environmental samples and other applications not listed above.  Samples can be analyzed by conventional-scale LC, coupled to the high-resolution mass spectrometer (Orbitrap Lumos).  Different chromatographic modes (e.g. RP, HILIC) are possible.

The following service is provided:

Service Fees Sample Requirements
LC-HRMS analysis $50/run Desalted samples free of large molecules

Other Applications

Please contact the facility for applications not listed here.

Private (for-profit) clients

Fees are double those listed above, for private sector clients.