Peptide Mass Fingerprinting (PMF) has been a cornerstone proteomic method from its inception. Proteins to be identified are enzymatically digested (usually by trypsin due to the predictable cleavage patterns) and the fragments are detected by mass spectrometry. Laser desorption techniques, such as Surface-Enhanced-Laser-Desorption-Ionization Time-Of-Flight Mass Spectrometry (SELDI-TOF-MS), generally provide clean, monoprotic digest peaks that can be used much like a fingerprint for protein analysis. Each mass peak generally corresponds to one of the digest’s peptide masses and thus, there is typically a ‘one peak to one peptide fragment’ spectra generated. This definition oversimplifies the actual results, however, this type of mass detection can be much cleaner (i.e. easier to interpret) than the data that typically comes from electrospray ionization (LC/ESI-MS). One of the major drawbacks from using laser desorption techniques, such as SELDI, comes from the energy absorption molecules (matrix) generating low-molecular-weight noise which can hide lower molecular weight peptide masses and, in turn, limits sequence coverage of the protein digest. Ciphergen has produced a way to conceptually reduce the matrix-generated noise and improve sequence coverage, while enhancing overall sensitivity. This technology is marketed as the SEND-ID ProteinChip Array (Cat. Number C573-0081 for the 8-spot array) and has the Energy Absorbing Matrix (EAM, α-cyano-4-hydroxy-cinnamic acid) already incorporated to the analysis target.
The SEND-ID ProteinChips come in a pack of 12 in a disposable Bioprocessor reservoir that can be quickly discarded since larger sample volumes are not recommended. The ProteinChips can be stored at room temperature and should be protected from light. The ProteinChip surface has a C18 functional group immobilized to it, thus, ‘dirty samples’ containing urea and salts can be directly applied and subsequently cleaned-up. Only a few micro-liters is required (<5uL) per sample making this, and all ProteinChips, attractive as a screening tool. A protocol is provided that outlines two processes, one for pure samples and one for not-so-pure samples. The sample must be mixed in 50% acetonitrile/0.2% TFA (pH <2) at a 1:1 ratio before adding it to the ProteinChip spot. Of course doing so puts any labile functional groups (ex. phosphate groups, cysteine bridges, hydrogen bonds, etc.) at risk thus limiting the information gained. After sample application, the sample is either dried as-is (for clean samples) or incubated on-target (‘dirty’ samples with urea, salts, etc.) for 10 minutes followed by an acid-wash (0.1% TFA that should be removed within 30 seconds, then the samples are washed again with ACN/TFA and allowed to dry. After the dry-down, analysis can begin making this a relatively quick and repeatable screening method.
The spectra gained are very clean, as advertised. Ciphergen recommends using lower laser energies and higher sample concentrations to further reduce noise-related signals. Overall a rather large improvement is observed when compared to typical MALDI/SELDI PMF analyses. I would recommend investing in this technology as another tool option for quick protein-digest analysis for any laboratory with SELDI-TOF capabilities interested in protein science.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research