Sequence information, from electrospray

J. A. Loo, C. G. Edmonds, and R. D. Smith. Primary Sequence Information from Intact Proteins by Electrospray Ionization Tandem Mass Spectrometry. Science, 248(1990) 201-204. 

Mass spectrometry is one of the major techniques in the interdisciplinary field of proteomics. It provides a rapid, sensitive and reliable means of protein identification and structural determination, allowing for development in this newly baptised but yet classical field of biochemistry and biomedicine. The use of electrospray ionisation in conjunction with a tandem mass spectrometer (MS/MS) provides essential amino acid sequence information from the m/z values of the so-called b andy ions formed from cleavage of the amide bond of a protonated peptide. This reaction requires proton catalysis, and the mechanism is of interest in the present context, since it is closely related to the processes occurring in other protonated carboxylic acid derivatives. 

Figure 2. Workflow of an LC-MS/MS experiment. A mixture of peptides from a protein sample digest is separated by reversed-phase chromatography on a nano-flow HPLC. The peptides elute from the RP column and are ionized by an electrospray source. In the first stage of mass spectrometry, m/z values and charge states for each precursor ion are determined and the most abundant precursor ions are selected for analysis in the second stage. The ions are then fragmented with by collision-induced dissociation (CID) a gas to produce fragment ions which are detected. Using the mass (from MS-1) and sequence information (from MS-2) protein sequence databases are searched to provide peptide identifications and protein matches.

Loo, J. A. Edmonds, C. G. Smith, R. D. Primary sequence information from intact proteins by electrospray ionization tandem mass spectrometry. Science 1990, 248, 201-204. 

The TIC trace from this analysis, shown in Figure 5.5, exhibits a maximum at ca. 19 min, and a representative electrospray spectrum is illustrated in Figure 5.6. Transformation of the latter produces the spectrum presented in Figure 5.7 which indicates the presence of two species with relative molecular masses (RMMs) of 56 548.5 and 58 161.4 Da. These masses are lower than the value of 59.1 kDa calculated from previously obtained sequence information. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

Electrospray ionization (ESI) is one of the two newer ionization techniques (the other being MALDI) that has revolutionized the application of mass spectrometry to biochemistry and molecular biology, and may fairly be said to have revolutionized these disciphnes also. This is the result of the ability of ESI-MS to provide molecular mass information of hitherto unthinkable accuracy and precision for fragile biopolymers, particularly proteins, and to provide amino acid sequence information for specific peptides present at trace levels in complex mixtures characteristic of biological extracts. The discipline of proteomics would not exist without the development of ESI and MALDI. However, this book is concerned with quantitation of small molecules present at trace levels in complex matrices, rather than the essentially qualitative data typically acquired in proteomics experiments, although application of the approach to quantitation of proteins and peptides is discussed in Section 11.6. Nonetheless, development of methods to produce and characterize gaseous ions from macromolecules was very important in the development of ESI-MS, and this history will be briefly described here. Excellent reviews of this history as it pertains to macromolecule characterization (Penn 1990 Smith 1991, 1992 Fernandez de la Mora 1992) are available, while... 

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

More extensive structural information is obtained when using MS-MS, where information on the peptide sequence can be extracted from the fragmentation pattern. So far, CE-MS-MS has predominantly been performed with electrospray ionization (ESI)-triple quadrupole and ESI-ion trap instruments, as reviewed in Ref. 13. The advent of ESI-TOF and ESI-quadru-pole-TOF instruments is believed to have a strong impact on CE-MS. TOF instruments require an extremely short time to produce a full mass spectrum and are especially attractive as a detection device for a separation technique producing sharp peaks, as illustrated by the separation of three enkephalins in a time window of 6 s with detection by means of ESI-TOF [14],... 

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