Mass Spectrometric Analysis of Peptides and Proteins
Triple Quadrupole Electrospray Ionization Mass Spectrometry
Electrospray ionization is one of the “softest” forms of ionization providing high sensitivity and is directly applicable to the analysis of peptides and proteins. Multiple charging through the use of electrospray ionization is sample dependent, but can extend the upper mass limit of the triple quadrupole instrument into the 50-80kDa range for some proteins. For mass spectral analysis of peptides and proteins, the sample must be soluble in dilute acid and it is recommended the sample undergo at least one round of hplc. A variety of information about the sample can be obtained through the use of electrospray-mass spectrometry on a triple quadrupole instrument. These include molecular mass measurement, assessment of chemical modifications through mass increases, daughter ion scans (for sequence analysis), precursor ion scans, constant mass difference scans and selected ion monitoring. Depending upon the sample and its ability to ionize, non-covalent associations may also be investigated.
Triple quadrupole technology enables one to select a peptide ion of interest and perform collision induced dissociation (CID) of the ion for sequence/structural information when sequence analysis cannot be obtained through traditional methods due to low sample amount. The maximum number of residues that can be sequenced is usually around 20 amino acids. Sequence analysis from larger peptides/proteins usually requires a digestion step before analysis. The Mass Spectrometry Facility offers in-gel digestion procedures on Coomassie or silver stained gels, solution digests, as well as digests from PVDF membranes which utilizes both triple quadrupole and MALDI-TOF technology (see below).
MALDI-TOF Mass Spectrometry
The mass spectrometry core also houses an Applied Biosystems Voyager-STR MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometer. This instrument extends the detectable mass range of various biomoleucles and polymers up to 400 kDa. The instrument is capable of analyzing positive or negative ions in the linear or reflectron mode. The latter mode provides high mass accuracy and the capability of performing post source decay (PSD) analysis for sequence/structural information. It is equipped with a 100 well sample plate and can perform fully automated acquisitions and data processing providing higher throughput.
Mass Profile Fingerprinting
Upon digestion of a protein with a specific protease, a set of peptide mass ions are obtained from the cleavage products, that are characteristic of that particular protein. Databases (such as MS-Fit, PepFrag and or Peptide Search) can then be searched, which try to “fit” the mass profile fingerprint with known proteins. Programs are also available at the facility to “digest” known protein sequences (with a variety of proteases) to predict theoretical peptide digest mass ions. Programs are also available (MS-Tag) for collision-induced-dissociation (CID, daughter ion analysis) data, which searches protein/peptide databases to try to “fit” the experimental daughter ion (CID) spectrum to masses of peptide daughter ions of a protein database to aid in sequence determination. Using expressed sequence tags (EST), only a portion of the ms/ms data is used to search the database for peptide/protein identification.
Mass profile fingerprinting procedures are performed using data obtained with the triple quadrupole and MALDI-TOF instruments.
Peptide sequence analysis by expressed sequence tags
The protein of interested was cut from a 1-D polyacrylamide gel and subjected to overnight in-gel digestion. Extracted peptides were desalted and concentrated on a microcapillary hplc C18 column and eluted directly into the mass spectrometer. A peptide molecular ion, (M+H)+ , m/z 843, was selected and subjected to collision induced dissociation. A doubly charged ion at m/z 421.83 is noted. In addition, three strong daughter ions at m/z 743.66, 672.34 and 570.81 are also noted.These were determined to be y ions of the following sequence:
Database searching by expressed sequence tags indicated that this peptide was obtained from a trypsin fragment VATVSLPR.
Microcapillary HPLC-Nanospray Mass Spectrometry
Our instrumentation is also fitted with a nanospray source. In conjunction with this source, we have developed an on-line hplc microcapillary system to interface with our triple quadrupole instrument. This type of technology extends the sensitivity of the instrument by allowing on-line concentration of the sample, increasing the signal to noise ratio. Microcapillary columns are custom-packed in-house to provide the researcher with a variety of stationary phases. Gradients are run on the microcapillary column in conjunction with a Rainin HPLC system, capable of providing preparative (up to 10ml/min), semi-preparative, analytical and narrowbore capabilities for off-line purposes. For on-line microcapillary-hplc, the output flow from the Rainin hplc system (400 ul/min) is split via an Accurate Microflow processor to flow rates down as low as 180-200 nl/min. Flow rates as high as 1000 nl/min can also be obtained. These flow rates are optimized for the 75um i.d. microcapillary columns. This application is currently being used for sequence analysis low level amounts of peptides/proteins.
In addition to the on-line nanoflow capabilities, the nanospray source also offers a nanovial option whereby small amounts of sample (1-5ul) can be analyzed which provides 15min-2h of analysis time from this small volume of material. This application is extremely useful in optimizing instrument conditions for MS/MS analyses and allows analysis of material from a limited volume.
Precursor Ion Scanning for Phosphopeptides
Triple quadrupole instrumentation allows unique capabilities in phosphopeptide analysis. Phosphorylated peptides (or phosphorylated peptides from proteins once digested), can be analyzed by electrospray ionization mass spectrometry in both the positive and negative ion mode. Fragmentation of the phosphorylated species in the negative ion mode provides a “unique” daughter ion (m/z 79) which is characteristic of a phosphate group. A precursor ion scan identifies those parent masses that yield the daughter ion with a m/z of 79 Da (PO3-), ie, only phosphopeptides. In the case where the protein sequence is known, the peptide molecular weights obtained are sufficient to identify the sequences that are phosphorylated. If the sample is an unknown sequence, CID spectra can provide potential sequence data including the site(s) of phosphorylation without the use of 32P.
Mass Spectrometric Analysis of Lipids
For years lipid analysis has been hampered by extensive chemical derivitization and purification prior to analysis. We have developed a mass spectrometric method whereby lipids can be analyzed by direct infusion into the mass spectrometer. Specifically, the Facility has been engaged in optimizing lipid ( both phospholipid and neutral lipid) extraction from whole cells. Analysis in the both the positive and negative ion mode will allow identification of the major phospholipid classes using traditional, parent ion and neutral loss scanning modes. Using this technology, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI) and its phosphates, phosphatidylglycerol (PG), phosphatidic acid (PA), and their plasmalogen analogues and ceramides can be detected. In addition, while CID in the positive ion mode yields information regarding head group analysis, CID in the negative ion mode yields information regarding the length of both fatty acid chains and the relative degrees of desaturation. Quantitation can also be performed in any of these analyses. Studies of importance utilizing this novel approach to lipid analysis include: 1) apoptosis, 2) liposome technology, 3)signaling molecules such as phospho- and neutral lipids and 4)glycolipids presented by the CD1 antigen. We have also extended our analysis of lipids by mass spectrometry to include mitochondrial, golgi and plasma membrane compartments.
Structural analysis of a phospholipid from Jurkat cells (m/z 885.38)
Lipids were extracted from Jurkat cells and a mass spectrometric lipid scan was performed. We were interested in determining the structure of the 885.38 mass ion. This ion was selected and subjected to collision induced dissociation. Mass analysis indicated that this was a phosphatidylinositol phospholipid with C18:0 and C20:4 fatty acyl chains running at m/z 283 and m/z 303, respectively. Further fragmentation of the inositol head group is also noted.
