GeLC-MS/MS express

The GeLC-MS/MS express service enables identification of proteins in gel bands present at low to sub-nanogram quantities. Protein which can be visualized by Coomassie brilliant blue staining or silver staining can almost always be positively identified by GeLC-MS/MS. Turn-around time is about one week for small sample sets. Please inquire for larger sample sets. Starting from $100 per sample.

GeLC-MS/MS Service

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Pricing:
$250 each, $200 for 8 or more, $150 for 24 or more, $100 for 48 or more
Assay:
GeLC-MS/MS Protein Identification 10k
Specifications:
UPLC-MS/MS, up to 10,000 sequencing events, 15 cm column, 20 min gradient
Sample types:
Any acrylamide-based gel spot or band

Sample preparation best practices

See our Guide to Handling Protein Gels and our Gel Staining Protocols.

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 is about one week.

Sample Preparation

UPLC-MS/MS

UPLC:
Thermo Easy-nLC 1000
Column:
C18 reversed phase 10 cm (length) by 75 micron (inner diameter) with integrated nanoelectrospray tip, heated to 50 C
Gradient:
20 min at 300 nL/min
Source:
Thermo Easy Spray
MS/MS:
Thermo Q-Exactive quadrupole-Orbitrap mass spectrometer

Data Analysis

Deliverables

FAQ

All Coomassie dyes and silver stains marketed as “MS-compatible” will work. Non-“MS-compatible” silver stains will also work, though there can be a decrease in sensitivity. Let us know if you have used a non-compatible silver stain and we will take steps to enhance protein recovery.

Bands which can be visualized by Coomassie Brilliant Blue stain are almost always identifiable. Bands visualized by silver stain are usually identifiable. The exceptions are typically very low abundance, low molecular weight proteins near the limit of detection of the silver stain and with few observable peptides. A Western blot is not a good indicator of suitability for LC-MS/MS.

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.

Some MALDI-TOF/TOF instruments do have excellent sensitivity (the Q-Exactive has sub-femtomole LOD). For MALDI-based analyses the peptides resulting from the in-gel digestion are typically spotted to a single target well on the MADLI plate. The entire spot is then ionized for TOF/TOF analysis. This means that the ionization energy is divided between all of the peptides in the sample and the detector has to attempt to sequence all the peptides on a very limited time scale - on the order of seconds. For LC-ESI-based analyses the peptides are chromatographically resolved (separated from each other) over the course of the gradient. The benefit of this is three-fold:

  1. the peptides do not compete for ionization energy with each other unless they co-elute
  2. relative sensitivity is improved because the peptides are not all competing with each other for detection at the same instant in time
  3. the mass spectrometer has much more time to sequence the peptides

Perform a very short-run SDS-PAGE separation:

  1. Obtain a pre-stained molecular weight ladder standard such as Invitrogen’s SeeBlue.
  2. Load your sample to one lane of a 16% SDS-PAGE gel and the pre-stained standard to another lane.
  3. Electrophorese at 100 V. Stop the electrophoresis as soon as your sample fully enters the gel.
  4. Remove the gel and then the gel cover plate. Do not remove the gel from the glass backing, do not rinse or stain the gel. Excise immediately with a clean scalpel to avoid contamination. Excise your sample from the top of the sample lane (the bottom of the well) down to a point level with the migration of the bottom-most marker of the pre-stained ladder. The band should not be more than about 5 mm in length.
  5. Place the excised band in a clean microcentrifuge tube pre-rinsed with lab-grade water.
  6. Fix the gel. Store at 4 C prior to shipping. Ship at ambient temperature.