Femtomole level

Our initial attempts with this design using the NDA/CN" system were encouraging far beyond our expectations. Our enthusiasm was sustained when we found that dipeptide-CBI derivatives could be detected at the femtomole level (Tables IV and V) and that such limits of detection with our purified derivatives under less-than-... 

Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B., Heck, A.J. (2004).. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943. 

The ProteinChip System from Ciphergen Biosystems uses patented SELDI (Surface-Enhanced Laser Desorption/Ionization) ProteinChip technology to rapidly perform the separation, detection, and analysis of proteins at the femtomole level directly from biological samples. ProteinChip Systems use ProteinChip Arrays which contain chemically (cationic, anionic, hydrophobic, hydrophilic, etc.) or biochemically (antibody, receptor, DNA, etc.) treated surfaces for specific interaction with proteins of interest. Selected washes create on-chip, high-resolution protein maps. This protein mass profile, or reten-tate map of the proteins bound to each of the ProteinChip Array surfaces, is quantitatively detected in minutes by the ProteinChip Reader. 

Femtomol levels of detection limits were also achieved in the determination of stimulant amines with the benzofurazan derivative 4-(Af,Af-dimethylaminosul-phonyl)-7-fluoro-2,l,3-benzoxadizole (DBD-F) [73], DBD-F was successfully applied to the PO-CL detection of amino acids and epinephrine [74] and a P-blocker, metoprolol [75], 4-(Af,Af-Dimethylaminosulphonyl)-7-hydrazino-2,l,3-benzoxadizole (DBD-H) has also been used for PO-CL determination of a neuronal cell protective compound, propentofylline. The method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus [85],... 

Examples are given in References 249 and 250 of about 100 ions detected in a single scan. This is about the practical detection limit for image current detection due to thermal noise in the detection system. Bradykinin has been detected from a sample concentration of 3 nM [249] and detection of sub-femtomole levels on a column is readily obtained [251]. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

Strobel, F.H. Solouki, T. White, M.A. Russell, D.H. Detection of Femtomole and Sub-Femtomole Levels of Peptides by Tandem Magnetic Sector/Reflectron TOF Mass Spectrometry and MALDI. J. Am. Soc. Mass Spectrom. 1991,2, 91-94. 

Durden DA, Boulton AA. 1988. Analysis of tryptamine at the femtomole level in tissue using negative ion chemical ionization gas chromatography-mass spectrometry. J Chromatogr 440 253. 

The most important advantage of OPA over other derivatization reagents is that it quickly reacts with amines and enables the BAs to be detected at femtomole levels. The drawback is that OPA reacts only with primary amines and leads to poorly stable compounds (120). Better results are obtained with DNS that also reacts with secondary amines and gives stable compounds that can be treated in subsequent concentration steps without using an automated system (103,121,5). 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Gatlin, C. L. Kleemann, G. R. Hays, L. G. Link, A. J. Yates, J. R. 1998. Protein identification at the low femtomole level from silver-stained gels using a fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem., 263, 93-101. 

Increase in sensitivity and efficiency of analysis in structural studies of enzymes with a gas phase sequencer have made it possible to determine the primary structure in a shorter period of time with a small amount of enzyme at the picomole or even femtomole level. In addition, thanks to the DNA sequencing technique the number of enzymes (or proteins) whose amino acid sequences are registered in a data base file has expanded explosively. The introduction of mass spectrometry on the primary structure determination of protein has stimulated the search for a new methodology other than Edman chemistry. 

Major histocompatibility complex (MHC) proteins are essential components of the immune system (1). One speeific role is for them to bind and present cellularly derived peptides (-8-10 amino acids - MHC Class I peptides) at the cell surface. These peptides are subsequently challenged by cytolytic T-lymphocytes (CTL s) which are programmed to differentiate between self and exogenous peptides. T-cell recognition of these latter peptides initiates a response that ultimately results in cell lysis and death of the infected cell. Hence, structural characterization of such peptides could potentially result in the development of therapeutie treatments of a number of infectious disease states such as viral cancers, AIDS, and autoimmune disease. However, the task of sequencing such peptides is difficult since MHC class I proteins can bind and present 10,000-15,000 different cellularly derived peptides present at the sub-pieo-femtomole level (2,3). 

A.L. McConnack, D.M. Schieltz, B. Goode, S. Yang, G. Barnes, D. Dmbin, J.R. Yates, III, Direct analysis and identification of proteins in mixtures by LC-MS-MS and database searching at the lorw-femtomole level. Anal. Chem., 69 (1997) 767. 

Various methods were evaluated for the targeted proteomics of human growth hormone (hGH) in human plasma [111]. hGH was spiked in plasma 10-fold above natural level ( 16 pg/pl). Iiutially, the full plasma proteome was reduced, alkylated, and digested prior to LC-MS via DDA on an ion-trap instrument. hGH could be identified from its T, peptide. Next, the plasma proteome was fractionated by RPLC and GE prior to digestion and LC-MS analysis. hGH could be identified with higher confidence. Finally, an LCxLC-MS approach was apphed, which enabled hGH identification from five tryptic peptides. An important conclusion was that hGH could be detected in a complex sample at the low femtomole level among proteins that were 40,000 x more abundant. The results show that a multidimensional approach may be taken for targeted proteomics and quantitative protein bioanalysis. 

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