Molecular Biology & Proteomics Core Facilty Dartmouth College

Protein Expression General Information

Protein Identification
Instrumentation
Static Nanospray
Search Engines

Protein Identification

We anticipate that most investigators’ primary interest will be the identification of proteins from 1D and 2D gels. Accordingly, this document and sample preparation instructions are directed at this method of protein identification. However, the primary process of digestion, fractionation and MS analysis has multiple entry points and will accommodate a variety of sample types. Fractions from liquid chromatography separations and immuno-precipitates after appropriate treatment are also commonly submitted for protein identification. If you are interested in or would like more information about these and other assays not covered on this website please contact the Proteomics Core facility. It cannot be stressed enough that sample preparation is the most crucial step in the process of protein identification. Investigators must perform a large part of the sample preparation in their own laboratories. If they lack access to specific pieces of equipment or have questions about their sample preparation we advise meeting with the core facility staff and or our faculty director Scott Gerber.

The key to protein identification using mass spectroscopy is the ability of our database search engines to match the mass (MS) and fragmentation pattern (MS/MS) of proteolytic fragments (peptides) observed in the laboratory with those generated in-silico from a database of known proteins. A crucial step in this process is the chemical or enzymatic digestion of the sample to produce peptides with predictable characteristics. Most commonly the protein is digested while it is still in the gel slice and the resultant peptides extracted from the slice. Digestion may be preceded by reduction of the protein’s disulfide bonds followed by alkylation of the sulfhydryl groups to prevent further reaction. Better results are obtained if alkylation and reduction are performed before the protein is run on the gel. This technique typically results in a large number of peptides with free amino termini and lysine or arginine at the C-terminal. Peptides from a tryptic digest can be directly analyzed by LC-MS/MS. For consistency the in-gel digestion needs to be performed in the Core Facility, this assures that all reagents, enzymes and process times follow accepted standards. The Core Facility provides a positive control. A negative control, a blank piece of gel, is provided by the investigator to track common contaminants typically keratin. The core facility monitors the results from our positive control sample batch to sample batch to insure consistency over time.

Instrumentation

The Core Facility purchased a ThermoFinnigan LTQ XL in 2006. The LTQ is an electrospray ionization linear ion trap mass spectrometer. The ThermoFinnigan LTQ XL is the mass spectrometer most used for protein identification from small amounts of sample. The electrospray interface allows samples to be directly introduced into the mass spectrometer. This has the advantage of allowing sample application in a variety of buffers and solvents.

As its name implies, ions formed during electrospray ionization are generated by a very small nozzle that produces micro-droplets. These droplets are desolvated resulting in the formation of lone charged peptide ions that are focused and eventually captured in an ion trap. The captured ions are then ejected from the trap and detected according to their m/z ratio. Because the ions are multiply-charged, only a limited m/z range is usually scanned, greatly increasing the mass accuracy. The greatest advantage of the LTQ is the ability to easily select an ion, fragment it and record the fragmented masses. Peptide ions can be trapped and further degraded (MS/MS). Peptides degrade in a predictable manner; m/z values can be found in the MS/MS spectra corresponding to the peptide and the peptide missing one, two, three etc. amino acids from both C and N termini. (See below.) The fragmentation gives rise to ions characteristic of the parent peptide ion’s composition and gives important primary structural/sequence information.

An additional advantage of this instrument is the ability to interface it with HPLC. A peptide mixture is partitioned on a column in one or two dimensions then directed into the ESI-IT instrument, allowing peptides to be analyzed as they are eluted from the LC column. It is important to understand the value of performing LC prior to mass analysis. LC not only separates the peptides it also concentrates them in a narrow peak. Concentration allows more peptide ions to be sprayed into the Mass Spectrometer greatly increasing sensitivity. This type of analysis can be performed on extremely small amounts of material; low femtomole amounts of peptide.

Static Nanospray

The Core Facility also has an Advion NanoMate source. The NanoMate allows prolonged spraying of a sample. This permits the operator to manually manipulate the conditions in the mass spectrometer to achieve the best spectra. This is a particularly effective method for analyzing synthetic peptides or other molecules that produce a characteristic fragmentation pattern.

Search Engines

The Core Facility uses two popular search engines for protein identification, Sequest and Mascot. Typically Sequest is used first, if the Sequest results are not satisfactory or show some ambiguity Mascot is then employed. More detailed information about Sequest or Mascot can be found on there respective websites.


				Protein Expression General Information Protein Identification Instrumentation Static Nanospray Search Engines Protein Identification We anticipate that most investigators’ primary interest will be the identification of proteins from 1D and 2D gels. Accordingly, this document and sample preparation instructions are directed at this method of protein identification. However, the primary process of digestion, fractionation and MS analysis has multiple entry points and will accommodate a variety of sample types. Fractions from liquid chromatography separations and immuno-precipitates after appropriate treatment are also commonly submitted for protein identification. If you are interested in or would like more information about these and other assays not covered on this website please contact the Proteomics Core facility. It cannot be stressed enough that sample preparation is the most crucial step in the process of protein identification. Investigators must perform a large part of the sample preparation in their own laboratories. If they lack access to specific pieces of equipment or have questions about their sample preparation we advise meeting with the core facility staff and or our faculty director Scott Gerber. The key to protein identification using mass spectroscopy is the ability of our database search engines to match the mass (MS) and fragmentation pattern (MS/MS) of proteolytic fragments (peptides) observed in the laboratory with those generated in-silico from a database of known proteins. A crucial step in this process is the chemical or enzymatic digestion of the sample to produce peptides with predictable characteristics. Most commonly the protein is digested while it is still in the gel slice and the resultant peptides extracted from the slice. Digestion may be preceded by reduction of the protein’s disulfide bonds followed by alkylation of the sulfhydryl groups to prevent further reaction. Better results are obtained if alkylation and reduction are performed before the protein is run on the gel. This technique typically results in a large number of peptides with free amino termini and lysine or arginine at the C-terminal. Peptides from a tryptic digest can be directly analyzed by LC-MS/MS. For consistency the in-gel digestion needs to be performed in the Core Facility, this assures that all reagents, enzymes and process times follow accepted standards. The Core Facility provides a positive control. A negative control, a blank piece of gel, is provided by the investigator to track common contaminants typically keratin. The core facility monitors the results from our positive control sample batch to sample batch to insure consistency over time. Instrumentation The Core Facility purchased a ThermoFinnigan LTQ XL in 2006. The LTQ is an electrospray ionization linear ion trap mass spectrometer. The ThermoFinnigan LTQ XL is the mass spectrometer most used for protein identification from small amounts of sample. The electrospray interface allows samples to be directly introduced into the mass spectrometer. This has the advantage of allowing sample application in a variety of buffers and solvents. As its name implies, ions formed during electrospray ionization are generated by a very small nozzle that produces micro-droplets. These droplets are desolvated resulting in the formation of lone charged peptide ions that are focused and eventually captured in an ion trap. The captured ions are then ejected from the trap and detected according to their m/z ratio. Because the ions are multiply-charged, only a limited m/z range is usually scanned, greatly increasing the mass accuracy. The greatest advantage of the LTQ is the ability to easily select an ion, fragment it and record the fragmented masses. Peptide ions can be trapped and further degraded (MS/MS). Peptides degrade in a predictable manner; m/z values can be found in the MS/MS spectra corresponding to the peptide and the peptide missing one, two, three etc. amino acids from both C and N termini. (See below.) The fragmentation gives rise to ions characteristic of the parent peptide ion’s composition and gives important primary structural/sequence information. An additional advantage of this instrument is the ability to interface it with HPLC. A peptide mixture is partitioned on a column in one or two dimensions then directed into the ESI-IT instrument, allowing peptides to be analyzed as they are eluted from the LC column. It is important to understand the value of performing LC prior to mass analysis. LC not only separates the peptides it also concentrates them in a narrow peak. Concentration allows more peptide ions to be sprayed into the Mass Spectrometer greatly increasing sensitivity. This type of analysis can be performed on extremely small amounts of material; low femtomole amounts of peptide. Static Nanospray The Core Facility also has an Advion NanoMate source. The NanoMate allows prolonged spraying of a sample. This permits the operator to manually manipulate the conditions in the mass spectrometer to achieve the best spectra. This is a particularly effective method for analyzing synthetic peptides or other molecules that produce a characteristic fragmentation pattern. Search Engines The Core Facility uses two popular search engines for protein identification, Sequest and Mascot. Typically Sequest is used first, if the Sequest results are not satisfactory or show some ambiguity Mascot is then employed. More detailed information about Sequest or Mascot can be found on there respective websites.


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