Global Proteomic Profiling

Animation of Q-Exactive Orbitrap mass analyzer performing MS/MS
Image courtesy of Thermo Scientific. Full video

Global proteomic profiling is an unbiased, shotgun proteomic assay utilizing ultra-high pressure liquid chromatography coupled to a high resolution, high mass accuracy quadrupole-Orbitrap mass spectrometer (Thermo Q-Exactive).

The assay can handle nearly any type of protein-containing sample, from bacteria to plants and blood to bone. Proteins are identified and quantified in each sample, then compared sample to sample.

Services

Standard Profiling

We offer five different assay lengths for different applications:

Service 101 – $400 Add to cart

Assay:
Global Proteomic Profiling 10k
ID:
Up to 1,000 proteins
Specifications:
UPLC-MS/MS, up to 10,000 sequencing events, 15 cm column, 20 min gradient
Sample types:
Affinity-purified samples or characterization of individual proteins

Service 102 – $1,000 Add to cart

Assay:
Global Proteomic Profiling 50k
ID:
Up to 4,000 proteins
Specifications:
UPLC-MS/MS, up to 50,000 sequencing events, 50 cm column, 90 min gradient
Sample types:
Sub-cellular fractions, exosomes, prokaryotic organisms

Service 103 – $2,000 Add to cart

Assay:
Global Proteomic Profiling 150k
ID:
Up to 9,000 proteins
Specifications:
UPLC-MS/MS, up to 150,000 sequencing events, 50 cm column, 240 min gradient
Sample types:
Eukaryotic organisms, biofluid plasma/serum/urine/saliva profiling

Service 104 – $4,000 Add to cart

Assay:
Global Proteomic Profiling 450k
ID:
Up to 15,000 proteins
Specifications:
3 high pH fractions, UPLC-MS/MS, up to 450,000 sequencing events, 50 cm column, 3 x 240 min gradient
Sample types:
Ideal for host-pathogen profiling and microbiome profiling, and other samples consisting of a mixture of organisms

Service 105 – $10,000 Add to cart

Assay:
Global Proteomic Profiling 1850k Library Building
ID:
Up to all detectable proteins
Specifications:
Multi-enzyme digestion, 12 high pH or OGE-IEF fractions, UPLC-MS/MS, up to 1,850,000 sequencing events, 50 cm column, 12 x 240 min gradient
Sample types:
Ideal for building a comprehensive proteomic library of a single organism for maximum sequence coverage and post-translational modification characterization

Plasma Profiling

Service 111 – $1,500 Add to cart

Assay:
Plasma Proteomic Profiling 150k
ID:
Up to 1,500 proteins
Specifications:
High abundance protein depletion via Agilent MARS-6, UPLC-MS/MS, up to 150,000 sequencing events, 50 cm column, 240 min gradient
Sample types:
Human plasma or serum
Requirements:
20 microliters of frozen human plasma or serum

Service 112 – $2,500 Add to cart

Assay:
Plasma Microparticle Proteomic Profiling 150k
ID:
Up to 3,000 proteins
Specifications:
Microparticle enrichment, UPLC-MS/MS, up to 150,000 sequencing events, 50 cm column, 240 min gradient
Sample types:
Plasma
Requirements:
5 mL of frozen plasma

Exosome Profiling

Service 113 - $1,500 Add to cart

Assay:
Exosome Proteomic Profiling 50k
ID:
Up to 2,000 proteins
Specifications:
Exosome enrichment, UPLC-MS/MS, up to 50,000 sequencing events, 50 cm column, 90 min gradient
Sample types:
Cell culture media or biofluids
Requirements:
10 mL of cell culture media, saliva or urine, 5 mL plasma

Phosphopeptide Profiling

Service 114 - $2,700 Add to cart

Assay:
Phosphopeptide Profiling 150k
ID:
Up to 5,000 phosphopeptides
Specifications:
Fe_IMAC column-based enrichment, UPLC-MS/MS, up to 150,000 sequencing events, 50 cm column, 240 min gradient
Sample types:
Any
Requirements:
1-10 mg of total protein

Workflow

Samples are processed upon arrival and placed in a queue for UPLC-MS/MS. Samples are analyzed on a first-come, first-served basis. Typical turn-around time for small sample sets is two to three weeks. Samples undergoing custom preparations, utilizing custom LC-MS methods and/or data analyses may require additional time. Please inquire for lead time on larger sample sets.

Sample Preparation

UPLC-MS/MS

UPLC:
Thermo Easy-nLC 1000
Column:
C18 reversed phase 50 cm (length) by 75 micron (inner diameter) with integrated nanoelectrospray tip, heated to 50 C
Gradient:
determined by assay (20 min - 4 hours) at 300 nL/min
Source:
Thermo Easy Spray
MS/MS:
Thermo Q-Exactive quadrupole-Orbitrap mass spectrometer

Data Analysis

Deliverables

Example Data Outputs

Example of a 4 hour UPLC-MS/MS assay. Total ion current chromatogram (top) and base peak chromatogram (bottom)

Examine tandem mass spectra and peptide assignments for every identification*

FAQ

A few micrograms of peptide digest is typically loaded for an assay. To account for sample losses and to permit re-analysis if necessary, we request about 50 micrograms of total protein per assay. Good results can be obtained from trace sample amounts - we can identify over 1,000 proteins from less than 1,000 cells procured via laser dissection.
Please send in the smallest volume possible. We will acetone precipitate protein in sample volumes greater than 2 mL.

Any species with an available sequence library (FASTA file) can be assayed. If a sequence library is not available for a species or if the library is limited in scope, we can attempt to search a library of related species to search for homologous proteins. There is no extra charge for searching a custom sequence library. We support all UniProt libraries by default. Search UniProt taxonomies to see if your organism is present.

In a typical complex sample such as a cell line we identify, on average, 5 different peptide sequences mapping to each protein and an average of 2 peptide spectral matches (identification events) per peptide.
The Q-Exactive has a sub-femtomole limit of detection (LOD). The LOD, limit of identification and limit of quantification for each peptide will vary based on several factors such as ionization efficiency and fragmentation characteristics.

Yes, we support SILAC and stable isotope dimethyl labeling. We recommend Life Tech reagents for SILAC experiments. We recommend this SILAC protocol and this stable isotope dimethyl labeling protocol

We prefer SILAC, but it is limited to cell culture experiments. SILAC controls for variability throughout the sample preparation and UPLC-MS/MS assay. In contrast, stable isotope dimethyl labeling can be applied to any sample type post-digestion. Because of this, it only controls for variability post-derivitization - primarily for UPLC-MS/MS.

Another option is to use a super-SILAC spike-in, in which a set of SILAC cultures are mixed to provide an adequate representation of the proteome. This super-SILAC mix is then mixed with the sample. This also controls for variability throughout sample preparation and UPLC-MS/MS.

We do not support isobaric tagging experiments (iTRAQ or TMT). We feel the instrumentation is not yet advanced enough to perform these assays without serious compromises which render this type of experiment less effective than label-free, SILAC or stable isotope dimethyl labeling experiments.

You will receive a free account on Proteome Cluster for viewing all details of the assays, including protein and peptide assignments, tandem mass spectra, run-level metrics, search parameters, etc. All data is available to download or share with your colleagues and is stored indefinitely.
Yes, we offer both iron- and titanium oxide-based phoshpopeptide enrichments. Due to the very low amount of phosphopeptides present in a sample, we recommend providing at least 1-10 mg of total protein. This will permit the identification of thousands of discrete phosphopeptides.

Yes, we offer albumin, immunoglobulin and multi-protein depletions from fluids such as plasma, urine and saliva.

For more infromation of sample-specific preparation considerations and shipping information see our guide.

Other questions? Please email support.

Further Reading

A few examples of from the scientific literature of leading research groups using the Q-Exactive.

Excerpt: “More than 2,500 proteins can be identified in standard 90 min gradients of tryptic digests of mammalian cell lysates - a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and MS/MS levels.”

http://dx.doi.org/10.1074/mcp.m111.011015

Excerpt: “Under the optimized experimental conditions, with a 50 cm long separation column and 4 h LC-MS run (including 3 h optimized gradient), 4,825 protein groups and 37,550 peptides were identified in a single run, and 5,354 protein groups and 56,390 peptides in a triplicate analysis of A375 human cell line, achieving ca. 50% coverage of the expressed proteome.”

http://dx.doi.org/10.1074/mcp.o113.028787

Excerpt: “Single runs… identified an average of 3923 proteins. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity.”

http://dx.doi.org/10.1074/mcp.m111.013722

Excerpt: “When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods…”

http://dx.doi.org/10.1074/mcp.o112.019802