Sample Requirements

The molecular weight of a protein can usually be obtained with a few picomoles by electrospray or laser desorption techniques if the protein has favorable solubility properties. Sample quantity requirements in these cases are usually dictated more by sample manipulation requirements than by the low femptomole actual sample consumption in the measurement. Hydrophobic proteins, particularly integral membrane proteins, present greater difficulty; however, we have developed methodology which permits analysis of intact integral membrane proteins by MALDI. Molecular weight measurement of smaller proteins and peptides (under ca. 2000 mass units) by FAB ionization can often be obtained with low picomole amounts of material. Larger species usually require larger amounts due to the greater difficulty of producing ions from larger molecules. Extremely hydrophobic or extremely hydrophilic molecules tend to be difficult to ionize by FAB, but the problem can often be overcome by forming a more favorable chemical derivative. ESI and MALDI generally require less sample. Tandem mass spectrometry requires significantly more sample than that required for molecular weight measurement. Present collision dissociation technology limits the ability to obtain complete sequences of peptides by tandem mass spectrometry to peptides under molecular weight of about 2500-3000 (approximately 25-30 amino acids). Partial sequence information can often be obtained, however, for larger peptides. With a peptide of molecular weight 2000 for which the molecular ion could be measured with picomole amounts, as much as two orders of magnitude more sample could be needed to obtain a good tandem mass spectrum.

Use of the array detector on the four sector instrument and use of the new ion trap instrument should significantly reduce these sample requirements. Since the current technology for tandem mass spectrometric sequencing requires peptide fragments of 25-30 amino acids or less, sequencing of a protein requires that it be cleaved into smaller fragments. The sample size requirement for sequencing a protein is therefore dictated not only by the mass spectrometry but also by the preceding peptide chemistry. Due to the practical aspects of sample manipulations in cleaving and isolating peptide fragments one should expect to require as much as nanomole to tens of nanomole amounts for small proteins in the 10-15,000 molecular weight range (tens to hundreds of micrograms). In theory a larger protein should not require any more sample on a molar basis, but the increasing complexity of the problem with larger proteins can lead to need for more sample.

It should be emphasized that while state of the art Edman sequencing can claim smaller sample requirements, the mass spectrometric methods do not require individual purification of each peptide fragment (which leads to a practical requirement for more sample). Furthermore, most Edman sequencing facilities do not operate at state of the art sensitivity for real samples. The above discussion of sample quantity requirements for tandem mass spectrometry relates primarily to sector type tandem instruments employing a point detector. The new scanning array detector on the MUSC four sector instrument is expected to be fully operational soon (11/96). Use of an array detector can increase sensitivity by as much as two orders of magnitude. The new (11/96) LCQ instrument is also expected to require significanly less sample. Two orders of magnitude improvement in instrument sensitivity does not necessarily directly translate to two orders of magnitude lower sample requirement, however, since sample manipulation limitations become more significant with extremely small samples. The answer to the question of how much sample is required is therefore not a firm figure. Every sample is a different molecular species with different properties, and the technology is constantly evolving. It can safely be said, however, that even with the current state of the art in protein and peptide mass spectrometry, the limitations lie more in the practical aspects of sample handling and peptide chemistry than with the mass spectrometry itself.

Another issue with respect to sample size requirement is that of the question being approached. For example, if the objective is to obtain some partial sequences for DNA probe construction, the sample requirement can be far less than would be required for full sequencing.

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We are located in room 305 of the Children's Research Institute Building at MUSC.

Biomolecular Mass Spectrometry Facility Department of Pharmacology Medical University of South Carolina 173 Ashley Avenue, CRI 305 Charleston, SC 29425	Telephone Numbers: 843-792-5849 (CRI 305) 843-792-2471 (department office) FAX: 843-792-2475