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Mass Spectrometry

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, Inc. employs highly experienced protein mass spectrometrists who are committed to providing data of the highest quality.

Protein Identification

NextGen Sciences offers several types of protein identification services:

MALDI Analysis

MALDI Analysis: An economical way to identify the most abundant one, or two, proteins in a solution or gel plug. Proteins that have been digested are identified using MALDI-TOF/MS analysis only.

General Protein Identification: When samples are received for general protein identification, we perform an in-gel/solution digest and attempt to identify the protein(s) using MALDI-TOF/MS. If successful, a report is issued immediately and you will be charged for the in-gel digestion and the protein id. However, if we are unable to conclusively identify the protein by MALDI-TOF, we proceed to the more sensitive technique of LC/MS/MS analysis at no additional cost. An in-house copy of the NCBI protein database is searched using the Mascot search engine. In the rare case that we cannot generate a list of tryptic peptides by either MALDI/MS or 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 NCBI protein database we offer de novo sequencing.

Extended Sequence Coverage Analysis: Using a combination of MALDI-TOF and LC/MS/MS analysis the sequence coverage can be extended beyond that of just one technique alone. When samples are received for extended sequence coverage analysis, we perform an in-gel/solution digest and attempt to identify the protein(s) using MALDI-TOF/MS. We then proceed to the more sensitive technique of LC/MS/MS analysis and combine both sets of data. An in-house copy of the NCBI protein database is searched and the protein ID’s are reported. In the rare case that we cannot generate a list of tryptic peptides by either MALDI/MS or 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 NCBI protein database we offer de novo sequencing.

Comprehensive LC/MS/MS Analysis: Our next level of service is aimed at identifying all detectable proteins from a solution or in-gel digest. This option requires the more sensitive LC/MS/MS analysis and therefore its cost is higher than general protein identification and extended sequence coverage analysis. All database searching is performed by one of our scientists and the service is not complete until we are satisfied we have identified every protein that is present in your sample at our detectable level and is present in the publicly available databases. Should you have access to proprietary databases, we can work with you to help you perform the search or allow us temporary access so that we can search the proprietary databases for you.

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Protein Characterization and De Novo Sequencing

De novo sequencing and protein characterization both entail manual interpretation of product ion data and, if necessary, sample manipulation (e.g. peptide derivatization) with further analyses to influence and allow rationalization of product ion data.

We typically utilize de novo sequencing when the source organism is poorly represented and distantly related to any other organism in the protein and EST databases. De novo sequencing can also be used to obtain peptide sequence information from novel proteins as well as non-tryptic peptides (such as naturally occurring peptides).

Protein characterization includes the identification of post-translationally modified peptides and localization of modification sites on the peptide. Due to their low abundance, only a few, highly-modified, peptides can be identified while performing routine protein identifications. In most cases, the identification of modified peptides requires additional sample preparation and manual data interpretation. Localization of modification sites additionally requires de novo sequencing of the modified peptide.

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Protein Spot Excision

Our laboratories use the Investigator™ ProPic robot to excise protein spots from polyacrylamide gels. The instrument can excise gel plugs from gels stained with fluorescent (Sypro® ruby) or visible (silver and coomassie) protein stains. The robot ensures accurate excision, enables optimized throughput of the proteomic experiment, and tracks the sample. The use of robotics for gel excision also minimizes keratin contamination which is particularly critical when pursuing the identification of low abundant proteins.

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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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MALDI/MS

MALDI/MS provides peptide mass fingerprinting for protein identification. Our spotting protocols have been optimized to enable automated MS acquisition which serves to improve spectra peak-picking algorithms. Data acquisition is performed on an Applied Biosystems Super-STR instrument and databases are searched using local copies of the ProFound search engine.

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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 a Micromass Q-Tof2 tandem mass spectrometer that we interface with a nano-LC system. Database searching is performed using Mascot.

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