Introduction

The Dartmouth Molecular Biology Core Facility has a Thermoquest LCQ Deca XP electrospray ionization-ion trap mass spectrometer (ESI-IT) and the ABI Voyager matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer. These instruments give the Dartmouth community up to date mass analysis capabilities, providing not only standard biomolecular mass analysis, but protein identification through sophisticated database searching capabilities. Although many interesting and sophisticated analyses can be performed with these instruments, we anticipate that most investigators’ primary interest in these new tools will be the identification of proteins from 1D and 2D gel bands. Accordingly, this document and the sample preparation instructions are directed at this method of protein identification. If you are interested in or would like more information about specific assays not covered in this document please contact the Molecular Biology Core facility. Please remember that we have limited personnel in our proteomics section at the present time. Users must perform a large part of the sample preparation in their own laboratories or, if they lack access to specific pieces of equipment, in the core facility with our guidance.

Background

Mass Spectrometry is a method for determining the mass of molecules by producing and analyzing charged species (ions), it measures the mass to charge ration (m/z) of ions. It is important to remember that mass is not directly measured it is derived from the m/z value. In the late 1980s and early 1990s improvements in ionization techniques facilitated the ionization of large biomolecules and the accurate mass determination of these species in the range of 0.01%-0.05%. Earlier techniques such as sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) produce a mass accuracy in the range ±5% and in some cases errors are much greater. Mass determinations are used in both the characterization and identification of biomolecules. Our mass spectrometers each have a different mechanism for creating ions and a different method for analyzing the ions. While the individual instruments have their own strengths and weaknesses, together they complement each other very well.

ABI Voyager DE Pro (MALDI-TOF)

The ABI Voyager DE Pro utilizes a laser whose output is matched to the absorption maxima of a matrix compound. The energy absorbed by the matrix results in a plume of volatile ions. If biomolecules are co-crystallized with the matrix they are also ionized in the plume. Ions from the plume are propelled down a long flight tube under the influence of an electric field. Since the time an ion takes to reach the detector at the end of the flight tube is inversely proportional to the mass to charge ratio (m/z), accurate measurements over a very wide range (>300,000 m/z) can be obtained. The advantages of MALDI-TOF include good sensitivity (into the femtomole range), and good tolerance to biochemical buffers and salts. In addition, since there is usually only a singly charged ion formed it is a good choice for the analysis of heterogeneous samples.

Access to our MALDI-TOF instrument is provided to qualified investigators who, after a short training session can use this instrument on a sign up basis.

Other uses of this instrument include obtaining molecular weights of purified molecules such as proteins, peptides, synthetic polymers and oligonucleotides. The instrument’s manufacturer, Applied Biosystems, provides a variety of “kits” for techniques such as c-terminal protein sequencing using a carboxypeptidase (subtractive) method. The availability of this kit is a good illustration of the accuracy of this instrument; to be able to identify an amino acid by the loss of its mass from a large protein is nothing short of spectacular. ICAT reagent kits are also available for quantitative determinations. See the Applied Biosystems web site for more information on these assays.

ThermoFinnigan LCQ Deca XP

ThermoFinnigan LCQ Deca XP is an electrospray ionization-ion trap mass spectrometer (ESI-IT). As its name implies ions formed during electrospray ionization are generated by a very small nozzle which produces micro-droplets. Under the influence of a potential and pressure gradient these droplets are desolvated resulting in the formation of multiply charged ions that are then 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. Advantages of ESI-IT are gentle ionization, the ability to adjust conditions to evaluate “native molecules” and in nanospray configuration it is more sensitive than MALDI-TOF.

ESI-IT can be coupled to HPLC (High performance Liquid Chromatography) and thus analyze complex mixtures such as peptide digests. This type of analysis is often referred to as LC-MS. However, one of greatest advantages of ESI-IT MS is the ability to easily select an ion, fragment it and reanalyze it. This is known as MS/MS and gives important structural/sequence information that can greatly aid identification. The disadvantages of ESI-IT include: interference by common salts and buffers, complex spectra, and inability to handle large molecules (m/z >2000Da) at the present time (enzymatically digested proteins result in peptides in this range). We have limited experience in the 2000-4000 m/z range, these procedures are still in the pilot study stage. Please inquire if you think the extra range is needed for your particular application.

Protein identification

In Gel Digestion
When using mass analysis to identify proteins, a crucial step in the process is the chemical or proteolytic digestion of the sample to produce peptides with predictable characteristics. Identification of a protein is made by comparing the mass of identified peptides to a database of peptides generated by a theoretical digestion of all known proteins. In gel tryptic digestion is a technique that was developed for the determination of primary amino acid sequence of N-terminally blocked proteins, but is commonly used for protein identification and proteomics. Digestion is usually preceded by reduction of the protein’s disulfide bonds followed by alkylation of the sulfhydryl groups to prevent further reaction. Most commonly trypsin is then used to digest the protein while it is still in the gel slice. This technique typically results in a number of peptides with free amino termini and a lysine or an arginine at the C-terminal. Peptides from a tryptic digest can be directly applied to a MALDI sample plate and/or can be subjected to RP-HPLC with the column effluent sprayed directly into the ESI-IT instrument. The in-gel digest is the most crucial step in the process of identifying in gel proteins, therefore this multi-step procedure needs to be performed in a prescribed manner with positive control provided by the core facility and a negative control provided by the investigator. It is also important for us to monitor the results from our positive control from sample batch to sample batch to insure consistency over time.

MALDI-TOF (ABI Voyager- DE-Pro)
The advantages of MALDI-TOF MS for peptide analysis and protein identification are speed, ease of analysis and accuracy of the molecular weights obtained by the TOF detector. The tryptic digest is spotted on the sample holder along with matrix (usually ?-cyano-4-hydroxycinnamic acid) dried, then placed in the instrument. A laser provides the energy to volatilize and ionize the peptides. A mixture of known peptides is placed on the sample holder near the sample for calibration purposes. Data from this known mixture are used to derive a calibration curve and the curve is then applied to the sample mixture. If there is sufficient high quality data the corrected peptide masses obtained from the sample are used to search databases. The assumption is that an individual protein will contain a distribution of peptide masses that will prove unique to a single protein in a database of virtually digested proteins. The NCBI non-redundant or the GenPep are examples of two such databases that are commonly used. A species specific database will be searched if one is available. Provided there is enough data (strong signals from a sufficient number of peptides) and the protein is in a database, this technique will identify a protein better then 90% of the time.

If there is not sufficient high quality data or the search does not yield and unambiguous identification additional sample can be used to obtain better data or further data manipulation and searching can be performed at additional cost. If it is determined that there is not sufficient material for a good MALDI spectra the investigator will be given the opportunity to submit the sample to LC-MS analysis on the ESI-IT.

ESI-IT (ThermoFinnigan LCQ Deca XP)
ESI-IT is the mass spectrometer used in most core facilities to obtain protein sequence data on very small amounts of sample. This type of analysis is more complex, more difficult and therefore more expensive than MALDI-TOF analysis.

The electrospray interface allows sample to be directly applied to the ionization chamber. This has the advantage of allowing sample application in a variety of buffers and solvents. A further advantage of this instrument is the ability to interface it with HPLC. The peptide mixture is partitioned on the column then directed into the ESI-IT instrument, allowing peptides to be analyzed as they are eluted from the LC column. The peptides can then 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. MS/MS provides important compositional data that is not obtained with MALDI-TOF. The accompanying Sequest software performs database searches by querying with not only the molecular weight of peptides, as with MALDI-TOF searches, but also queries with compositional data obtained by MS/MS. This analysis can be performed on extremely small amounts of material; low femtomole amounts of peptide, at least two orders of magnitude lower then the MALDI-TOF, can be detected. This is about the same level of detection as the most sensitive silver stained gel.

If your sample is determined to be more suitable for ESI-IT because of the expectations of the investigator or because it has been deemed unsuitable for MALDI analysis, it will come directly to this point after digestion. For this analysis the sample is applied to an RP-HPLC column and the column effluent directed through the nanospray source into the mass spectrometer. The mass spectrometer will perform MS and MS/MS of the HPLC separated peptides. A Sequest database search will be performed using the data obtained from these analyses. If the protein in question is not found in any of the available databases, de novo sequencing using the MS/MS data can be performed at extra cost and requires a good spectra for success.

De Novo sequencing can be performed on this instrument using the MS/MS data making it useful for obtaining primary amino acid sequence of proteins not found in databases. Some post-translational modifications can be located and analyzed by these methods as well. Multiple samples requiring digestion by enzymes with different AA specificity are often required for these kinds of analyses. Database searching with primary sequence data obtained by MS/MS also allows for the analysis of mixtures of proteins and is a very powerful technique. We are in process of developing the two dimensional chromatography system necessary for this analysis.