Mass spectroscopy: Mix and match

Proteomics technology: Character references I used to usher the proteins through the intermediate steps that separate these two techniques, date back to the 1960s. Most venerable of all is the century-old separations technique of chromatography. Fortunately for scientists aiming for widespread protein characterization in the wake of the triumph of genome sequencing, a series of improvements in mass spectrometry and 2D-gel technology is readying these tools for the task that lies ahead. Chromatography was modernized in the 1970s with the invention of highpressure pumps, the addition of multiple columns and improved packing materials for columns, leading to its modern incarnation as high-performance liquid chromatography, or HPLC, a workhorse in many life-sciences labs. Established scientific-equipment companies are also working to integrate more steps of the overall proteomics workflow into fewer pieces of equipment. And many start-up companies are looking for ways to enhance or supplant parts of the established proteomics process. ass spectrometry represents a worldwide market worth US$1 billion a year, with about a third of that dedicated to machines especially suited for proteomics. The system uses three components — an ionization source, an analyser and a detector. Users have at least two choices for each component. Assorted pairings offer different advantages — some combinations are more suited to proteomics, whereas others lend themselves more to small-molecule analysis. And some combinations will integrate with other proteomics equipment such as liquid chromatography. Companies are tending to make their new machines more versatile, more automated and more compatible with other proteomics equipment — but, in general, the more choices offered by one Integration: speeding analysis. NATURE | VOL 413 | 25 OCTOBER 2001 | www.nature.com GYROS The mass spectrometer is key to proteomics. Although there are many different methods emerging — from mapping all the proteins in a single organism to describing the multitude of interactions experienced by proteins during their lifespan — the general technique of isolating and identifying the many proteins in different cell types remains central. Mix and match MASS SPECTROMETRY M SPL n the Odyssey, Homer’s hero has his hands full when he faces Proteus. The demigod challenges Odysseus by transforming himself into a lion, a boar, a serpent, a wave and finally a tree. In proteomics, scientists trying to discern the nature of proteins face an equally formidable challenge, because protein data are as mutable as Proteus. Protein levels in different cell types change constantly as they are upregulated, downregulated, cleaved and phosphorylated. Because protein information, unlike DNA, is not static in the cell, scientists must follow Odysseus’ lead. They will have to be resourceful, especially as the tools used in today’s high-throughput environment still bear the stamp of an earlier era when one protein at a time was the standard. The 2D gel used to separate individual proteins from complex mixtures dates back to the mid-1970s. Mass spectrometry, which identifies proteins by weight once they are isolated, has been around since the First World War. And industrial robots, technology feature machine, the higher the price tag. The choices begin where the process starts — ionization sources. Ionization gives the sample an electric charge. The widely used MALDI (matrix-assisted laser-desorption/ ionization) uses solid samples, and produces ions of large and small molecules. Electrospray ionization (ESI) is used less often in proteomics. It ionizes liquid samples and is most often used for peptides and small molecules. It can be directly coupled to liquid chromatography systems. For analysis, time-of-flight (TOF) is most frequently used with MALDI, whereas ESI is usually coupled to quadrupole or ion-trap analysers. Quadrupole machines are considered lowperformance instruments compared with MALDI-TOF, but they only cost about a third as much. Ion-trap analysers are also modest performers, but they are robust and easier to look after than the other types, and are even more modestly priced. Finally, there are two kinds of mass spectrometer — MS and MS/MS. MS is the faster, easier-to-operate option. But, in addition to generating a spectrum of the sample, MS/MS can take some of the ions that have been separated and measured, fragment them further, and then generate spectra of those parts. This allows users to discern which amino acids the peptides contain, and, in some cases, can identify the sequence of these amino acids within the peptide. © 2001 Macmillan Magazines Ltd 869 Identifying spots on gels can be time consuming. he says. They are getting bigger, so more sample can be loaded, which improves the detection of low-abundance proteins. ‘Zoom’ gels have also been developed with ever-narrowing pH ranges, which give better resolution as well as higher sensitivity. Fluorescent labelling is also getting better, he says. Differential-expression analysis using difference gel electrophoresis, developed at Carnegie Mellon University, allows up to three samples to be run simultaneously on a single gel using cyanine-dye chemistry. This should let researchers detect protein differences between normal and cancerous tissues on the same gel. The method also allows multiplexing of gels, which significantly increases throughput, reproducibility and accuracy. Multiple gels provide comparative analysis and accurate measurement of differential protein expression. Although the handling and analysis of 2D gels have improved dramatically, Rodin notes that complementary techniques, such as X-ray crystallography, are needed to resolve the whole proteome. Fortunately, the next stage of the proteomics pipeline, handling the intermediate steps between electrophoresis and mass spectrometry, is becoming easier. Picking the protein spots off the gels, then digesting them into peptide fragments used to be two separate, manual tasks. Now they are becoming automated and are being integrated into the workflow (see ‘Multiple choice’, below). But improving and combining individual components can be challenging, says Steve Martin, director of Applied Biosystems’ Proteomics Research Center in Framingham, Massachusetts. For example, increasing the capacity of one instrument without accounting for the additional need for throughput in others can actually result in bottlenecks, he says. Three commercial — and by today’s standards, integrated — systems are made by Amersham Biosciences, Genomic Solutions in Ann Arbor, Michigan, and Bio-Rad in Hercules, California. Their basic components are similar — they all use robotic sample-preparation, 2D-gel electrophoresis, excision of spots, labelling, and ionization and analysis of the peptide fragments by mass spectrometry. In these systems, data generated from all the instruments are presented in a userfriendly graphical (...truncated)