General Technique

Currently, the most effective way to identify and quantify proteins and their modifications by mass spectrometry (MS) is to analyze peptide digests of the proteins of interest. This creates very complex mixtures of peptides. In order to comprehensively characterize complex mixtures of peptide, we are developing methods to better separate peptides prior to mass spectrometric analysis. These methods can be used in succession to achieve a very high degree of resolution. Furthermore, some of the methods provide information that can be used to discriminate correct from incorrect search results.

1) Reversed phase chromatography. Reversed phased chromatography is the most common fractionation technique used for separating peptides prior to mass spectrometry. In this technique, peptides stick to reverse phase resins in aqueous buffers, and they are eluted as the concentration of organic solvent increases. The resin is usually made up of hydrophobic alkyl chains ( -CH2-CH2-CH2-CH3 ) that interact with the peptide. Peptides are separated based on their hydrophobic character by running a gradient of the organic solvent. During electrospray ionization MS, microcapillary reversed phase columns are often operated in-line with the mass spectrometer.

2) Strong cation exchange (SCX) chromatography. SCX columns are packed with resin the carries a net negative charge at low pH. At low pH, tryptic peptides carry a net positive charge. In low salt conditions, positively charged tryptic peptides will bind to the SCX resin, and they can be eluted by running a gradient of increasing salt concentration. Peptides are separated based on charge and hydrophobicity. Fractions are collected and analyzed by MS.

3) Isoelectric focusing (IEF). The use of isoelectric focusing in proteomics is most often applied to separation of proteins, and it is often followed by SDS-polyacrylamide gel separation of the proteins by molecular weight. Institute for Systems Biology (ISB) researchers are exploring the use of this standard technology for the separation of peptides prior to microcapillary reversed phase liquid chromatography-mass spectrometry. Differences in peptides isoelectric points (pIs) are the basis of separations by isoelectric focusing. The pI is the pH at which a peptide carries a net charge of zero, and it is determined by the charged groups in the peptide. When the pH is equal to the pI of peptide, the peptide will not migrate in an electric field. For every peptide there is a specific pH at which its net charge is zero. When a peptide is placed in a medium with a pH gradient and subjected to an electric field it will initially move towards the electrode with the opposite charge. During migration through the pH gradient, the peptide will pick up or lose protons. As it migrates, the net charge and the mobility will decrease and the protein will slow down. Eventually, the peptide will arrive at the point in the pH gradient which is equal to its pI. Here it will be uncharged and stop migrating. In this way peptides are focused into sharp bands. They can then be extracted and analyzed by MS.

4) Free flow electrophoresis. Free Flow Electrophoresis (FFE) is an electrophoresis procedure working continuously in the absence of a stationary phase (or solid support material such as a gel). Like IEF, it separates charged particles, like peptides and proteins according to their pIs. Samples are injected continuously into a thin buffer film, flowing through a chamber formed by two narrowly spaced glass plates. Current is applied perpendicularly to the electrolyte and sample flow while the fluid is flowing (continuous FFE) or while the fluid flow is transiently stopped (interval FFE). The applied electric field leads to movement of charged sample components towards the respective counterelectrode according to their electrophoretic mobilities or isoelectric points. The sample and the electrolyte used for a separation enter the separation chamber at one end and the separated sample components are collected at the other side. After purifying the peptides away from electrolytes and other buffer components, the peptides can be analyzed by MS.

Compared to IEF, FFE is not limited by sample loadability. This is an important consideration for some large scale proteomics experiments. ISB researchers are also using FFE to fractionate proteins, macromolecular complexes and organelles.

5) Cysteine capture. Selective isolation of cysteine-containing peptides is an effective way of reducing sample complexity. Two strategies are commonly used at the ISB to isolate cyteinyl peptides. Both approaches take advantage of the specific reaction of iodoacetyl groups with thiols to form a covalent bond at pH 7.5-8.5. The ICAT reagent consists of an iodoacetamide group linked to a biotin moiety. After reaction of the iodoacetamide group with cysteinyl thiols, the cysteine-containing peptides are isolated via the biotin moiety by avidin affinity chromatography. In the solid phase approach, an iodoacetamide group is linked to a glass bead via an acid cleavable linker. Cysteine-containing peptides are immobilized by incubation with the solid phase reagent. Subsequent incubation in strong acid results in release of the cysteine-containing peptides. Cysteine content can be used as a powerful discriminator during the interpretation of MS data obtained on peptides isolated in this way.

6) Phosphopeptide capture. Identification of phosphopepetides is difficult because phosphopeptides ionize poorly, and they are often present in substoichiometric quantities. We have developed a procedure that consists of the following steps:

1. Peptide mixtures are methyl esterified by reaction of carboxylate groups with methanol. Blocking carboxylates prevent them from further reaction in subsequent steps.
2. Phosphate groups are activated using carbodiimide and imidazole, and reacted with excess immobilized polyamine to form phosphoramidate bonds.
3. Covalently bound phosphopeptides are eluted by acid hydrolysis of the phosphoramidate bond and recovered for MS analysis.

7) Glycocapture. We have developed a method for the isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides, and the specific release of formerly N-linked glycopeptides by peptide N-glycosidase F. The recovered peptides are then identified and quantified by mass spectrometry.

8} Gas phase fractionation (GPF). In this approach, the resolving power of the mass spectrometer is used to fractionate peptides based on their m/z ratio. In a typical MS analysis, the mass spectrometer is programmed to scan from 400-1800 m/z. In GPF, a peptide mixture is analyzed multiple times with the mass spectrometer programmed to analyze different narrow mass windows in each run (i.e., 400-800 m/z, 800-1200 m/z, 1200-1800 m/z). In this way, GPF can increase proteome coverage because it reduces sample complexity and gives the mass spectrometer more time to thoroughly analysis peptide ions eluting in each narrow mass window.

Purpose/use/application of the technique:

Fractionation of protein digests prior to mass spectrometry analysis increases data density (i.e., the number of peptides in complex samples that can be identified by MS). In particular, peptides of low-abundance or low-ionization efficiency can be readily identified due to the minimization of suppression of ionization by co-present peptides by prior fractionation. In addition, fractionation helps to overcome duty cycle limitations of some mass spectrometers.

Example(s) of projects at ISB that use this technique:

• Yu Lu - IEF
• Bernd Wollscheid, Andy Tao - phosphopeptides.
• Jeff Ranish- Cysteine capture
• Hui Zhang- Glycocapture
• Ming Li - FFE

Ongoing area of technology development:

We continue to improve the resolution and recovery these techniques

Representative publication(s):

(Lu et al., 2004) (Ideker et al., 2001; Yi et al., 2002; Zhang et al., 2003; Zhou et al., 2002; Zhou et al., 2001)

Ideker, T., Thorsson, V., Ranish, J. A., Christmas, R., Buhler, J., Eng, J. K., Bumgarner, R., Goodlett, D. R., Aebersold, R., and Hood, L. (2001). Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929-34.

Lu, Y., Bottari, P., Turecek, F., Aebersold, R., and Gelb, M. H. (2004). Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags. Anal Chem 76, 4104-11.

Yi, E. C., Marelli, M., Lee, H., Purvine, S. O., Aebersold, R., Aitchison, J. D., and Goodlett, D. R. (2002). Approaching complete peroxisome characterization by gas-phase fractionation. Electrophoresis 23, 3205-16.

Zhang, H., Li, X. J., Martin, D. B., and Aebersold, R. (2003). Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21, 660-6. Epub 2003 May 18.

Zhou, H., Ranish, J. A., Watts, J. D., and Aebersold, R. (2002). Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 20, 512-5.

Zhou, H., Watts, J. D., and Aebersold, R. (2001). A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19, 375-8.