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Overview

We offer multiple protein mass spectrometry services that are tailored to the individual project. Our mass spectrometry facility is housed in a HEPA filtered laboratory with stringent temperature and humidity control. This is important to control for airborne contaminants and for accurate instrument calibration, respectively. NextGen Sciences employs highly experienced protein mass spectrometrists who are committed to providing data of the highest quality.

• Protein Identification

Mass spectrometry is used to identify the proteins in a solution or in a gel plug (1D or 2D SDS-PAGE gel) using highly sensitive nano LC methods and LTQ Orbitrap XL instrumentation.

• Protein Characterization and De Novo Sequencing

Mass spectrometry is used to identify and localize post translational modifications to specific peptides and amino acids. De novo sequencing is performed for proteins that do not have sequenced genome information.

• Protein Quantitation

We offer multiple options for mass spectrometry based protein quantitation using labeled and non-labeled techniques.

• Protein Digestion


• Equipment

Protein Identification

Samples received for protein identification are analyzed using the sensitive technique of nano LC/MS/MS on either an LTQ or LTQ Orbitrap XL mass spectrometer. Typically, protein samples are first digested using trypsin to create a peptide mixture which are then injected on our custom-designed LC systems and eluted into the mass spectrometer which usually operates in data-dependent mode. Years of experience optimizing digestion, sample handling, chromatography and mass spectrometer conditions allow us to achieve unparalleled levels of sensitivity and coverage. Product ion data is searched in-house using forward and reversed database searching methods to allow assessment of false discovery rates. Search results are parsed into the Scaffold visualization software to allow validation of protein assignments. We give both tabulated reports and Scaffold files (accessible through a free viewer) to clients allowing for full inspection of the data.

In the rare case that we cannot generate a list of tryptic peptides by LC/MS/MS we will only charge you for the in-gel digestion. If a list of tryptic peptides is generated and it does not match to anything in the protein databases we offer de novo sequencing services.

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Protein Characterization and De Novo Sequencing

Protein characterization includes the identification of post-translationally modified peptides and localization of modification sites on the peptide. Often, due to their low abundance only a few modified peptides can be identified while performing routine protein identifications. Therefore, the identification of modified peptides can require additional sample preparation. The ability of software tools to confidently map modification sites is often limited and as such we perform manual data interpretation/validation to allow for complete confidence in the data.

De novo sequencing services are available when the source organism is poorly represented and distantly related to any other organism in the protein and EST databases. We also utilize de novo sequencing when working with novel proteins, endogenous peptides (such as naturally occurring peptides) or protein therapeutics.

Both protein characterization and de novo sequencing require high resolution and high mass accuracy product ion data and as such we exploit the LTQ Orbitrap XL for such projects.

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Protein Quantitation

See discoveryexpress™ for options available

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Protein Digestion

We perform all in-gel and solution digestion of proteins robotically using the Investigator™ ProGest. Our protocols have been developed to provide maximum recovery of peptides post-digestion, and to ensure that mass spectrometry- interfering substances are removed from the sample. Robotic digestion also minimizes the risk of keratin contamination and allows for high throughput sample processing.

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nanoLC/MS/MS

Protein identification and characterization using LC/MS/MS has become the workhorse of proteomics, primarily because complex mixtures of proteins can be analyzed (in-gel or in solution), high sensitivity levels are attainable and the sites/identity of post-translational modifications can be probed. The laboratory is equipped with the following:

• LTQ Orbitrap XL (ThermoFisher)
• TSQ Quantum Ultra (ThermoFisher)
• LTQ (ThermoFisher)
• Q-ToF2 (Waters)
• nano Acquity UPLC systems (Waters)

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