Created: 8th September 1998, last updated: 9th September 1998, © 1998 ABRF

This was the 10th in a series of symposia jointly sponsored by ABRF and ASBMB designed to bring to researchers in biochemistry and molecular biology the latest information on technologies used in their research fields. In addition to describing emerging technology, practical information on how the new techniques can be accessed and applied to their research problems was presented.

The Symposium was opened by Gary Siuzdak of The Scripps Research Institute. His presentation, "Finding a Mass Spectrometer to Meet Your Needs" described the following desired attributes for mass spectrometry instrumentation: femtomole sensitivity, LC/MS interface, ppm accuracy, high mass range, the ability to analyze small molecules, low cost, and small size. As he pointed out, no one instrument is capable of providing all of these features, however both electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are coming close to meeting the ideal system (Table 1).

Suizdak discussed the general utility of MALDI and ESI instruments for peptides and protein analysis. Some of their respective advantages include the ease of interfacing ESI with LC, as well as ESI sample preparation which may be easier for non-specialists to learn. He also discussed the superior capacity of MALDI to perform whole protein and simultaneous mixture analysis. It was pointed out that while both ESI and MALDI mass spectrometers are becoming easier to use, when buying a system it is important to focus on the strengths of each technique in terms of a lab’s individual needs.

The next presentation by Ron Niece, UC Irvine, focused on ABRF mass spectrometry data from member laboratories around the world. The incorporation of mass spectrometry into biomolecular resource laboratories has been dramatic over the course of the last decade because mass spectrometry is a technique that 1) is needed on occasion by many researchers, 2) requires expensive capital equipment, and 3) is expertise-intensive. In 1987, no resource laboratories surveyed (Williams, KR, et al., FASEB J. 2: 3124, 1988) indicated that they were offering mass spectrometry. In 1992, nearly one in eight offered the service and in 1996, nearly 50% of ABRF laboratories offered mass spectrometry capabilities (Niece, RL, et al., Association of Biomolecular Resource Facilities, in Encyclopedia of Bioprocess Technology, Flickinger, MC, and Drew SW, Eds, Wiley & Sons, in press). Preliminary data from the 1998 Survey committee indicated that MALDI is more readily available than ESI.

In his presentation on what it takes to be successful when analyzing proteins by mass spectrometry, Roland Annan of Smith Kline Beecham Pharmaceuticals discussed capabilities of mass spectrometry as it is used in their laboratory, noting that any well-trained person can obtain the basic information from either type of mass spectrometer, ESI or MALDI. Mass spectrometry is one of the analytical techniques they use because it: 1) provides an intrinsic property (mass-to-charge ratio), 2) furnishes molecular weight information of picomole to femtomole quantities of proteins and peptides, independent of covalent modifications, 3) may do this from mixtures without separation, and 4) permits acquisition of partial to complete sequence data.

The trade-off between mass accuracy and quantity of material available often determines whether ESI or MALDI is the preferred method for analysis. MALDI is often more sensitive (e.g., picomole amounts of sample at 0.1% accuracy) while ESI often provides better accuracy (e.g., 0.01 % accuracy on 100 picomoles of sample). Differences in mass from expected molecular weights provide information about the identity of synthetic peptides or modifications of proteins. Database searching on masses of peptides following digestion have built-in redundancy; this provides increased confidence of identity because more matches are observed and errors decrease (Table 2).

Catherine Castello of Boston University continued the discussion of proteins, pre and postranslationally modified. The focus was on the transthyretin protein in familial amyloidotic polyneuropathy. This protein is usually a covalently modified tetramer. Normal and mutant monomers are cysteinylated or phosphorylated. A mixture of these forms is found when purified from heterozygous individuals. Identifying the many postranslational modifications in collagen indicated several successive levels of modifications, such as glycosylation of modified proline and lysine residues. She also described the use of MALDI with an infrared laser ionization source that allows for deeper penetration into the matrix, less fragmentation, and higher resolution.

Robert McIver of UCI and IonSpec provided a forward-looking discussion on the use of FTMS as an ion detector. Fourier transform mass spectrometry can store ions from an external ion source for as long as hours in some cases. The long time—scale leads to a high signal-to-noise ratio which in turn improves resolution and sensitivity; resolution can exceed 400,000 and internal mass calibrations can lead to errors of only about 1 ppm. The high resolution allows for ESI charge states to be calculated directly from isotope peak spacing. McIver also discussed the utility of ESI FTMS for the analysis of non-covalent interactions.

In general, the applicability of mass spectrometry to biochemical problems is quite extraordinary and what is interesting about the technology is that it has, in many respects, exceeded the needs of biochemists. What we are currently observing is the biochemistry community gradually coming to learn of the potential of this technology. At the same time, mass spectrometry researchers are pushing the limits of the technologies’ capabilities even further (MSⁿ, laser photodissociation, hydrogen/deuterium exchange and non-covalent interaction). It is a beautiful time to be in mass spectrometry and an even better time to be a biochemist!

Table 1. General comparison of ionization sources.

	Electrospray Ionization (ESI)	NanoESI	MALDI
Typical Mass Range	70,000	70,000	300,000
Matrix Interference	none	none	yes
Degradation	none	none	possible photodegradation and matrix reactions
Ability to Analyze Complex Mixtures	somewhat limited	somewhat limited but better than ESI	excellent
LC/MS Capability	excellent	OK, but low flow rates can present a problem	very limited (off-line)
Sensitivity	high femtomole to low picomole	high zeptomole to low femtomole	low to high femtomole
Accuracy and MS/MS capabilities	mass analyzer dependent	mass analyzer dependent	mass analyzer dependent
Salt Tolerance	low (low millimolar)	moderate (low-mid millimolar)	moderate(low-mid millimolar)
Comments	Multiple charging useful. Significant suppression can occur with mixtures. Soft ionization = low fragmentation LC/MS easily automated and quantitative	Multiple charging useful, but significant suppression can occur with mixtures. Soft ionization = low fragmentation	Matrix background can be a problem for low mass ions. Soft ionization = low fragmentation

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Table 2 Comparison of number of peptide matches at various error levels. Database searched: EMBL-NonRedundant Protein Database (>230,000 sequences).

Mass List	Error	Result
1226.680	0.1%	>11,500 proteins
1226.680	0.1%	>21,000 have either
1429.756		1,180 have both
1226.680	0.1%	>2,985 have two/three
1429.756		192 have all three
1786.899
1226.680	0.1%	38 have all four
1429.756	0.01%	only 1 has all four
1786.899	0.001%	only 1 has three/four
1021.603

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