This Committee was formed in September 1996 and is the youngest of all Research Committees. Its first study included a survey that was sent to all ABRF member laboratories, and a test sample was provided to all laboratories requesting it.
Goals of the survey included determination of the status of mass spectrometry among ABRF membership in terms of types of instrumentation in use, level of experience and expertise, types of applications utilized, and techniques used. At the same time, the survey asked for input to determine the direction the Committee should take in designing future studies and in providing mass spectrometry related information to the membership.
The survey was returned by 38% of member laboratories of which nearly 90% do mass spectrometry and 67% offer it as a service. This represents 25% of the total ABRF membership. Most of the respondents (73%) have 1-10 years of experience in mass spectrometry, 14% have more than 10 years, and 13% have less than one year experience. Almost half the respondents use MALDI-TOF, and a third use electrospray quadrupoles. The remaining laboratories are using ion-trap instruments and other combinations of source and analyzer such as FAB, FTMS and magnetic sector. Of all respondents, 55% have only one instrument available; however, almost 8% of all laboratories have more than three instruments. The majority of all samples analyzed are proteins and peptides (69%), followed by carbohydrates and glycoproteins (14%), oligonucleotides (9%), lipids (3%), peptoids (2%), and peptidylnucleic acids (2%). Most laboratories obtain their samples as synthetic compounds and request 1-10 picomoles. Interestingly, 46% of the respondents are engaged in some kind of sequence analysis, which includes PSD, CAD, and ladder sequencing. Great interest was expressed in MS sequencing techniques, protein digestion and identification, and related forms of protein sequencing.
The goal of the test sample study was to address some basic issues such as resolution, accuracy, and the separation and identification of components in mixtures by mass spectrometry. Participants were also encouraged to utilize supporting techniques.
The test sample was designed to allow participation at all levels of expertise and technical capabilities. Participating laboratories were requested to perform the mass analysis on two samples, MS-1 and MS-2, with the highest possible resolution and accuracy. They were requested to calculate and report the resolution (FWHM method). All laboratories who felt they had the technical capabilities were invited to identify the components. MS-1 was porcine N-acetyl renin substrate (tetradecapeptide corresponding to the amino-terminus of angiotensinogen). MS-2 was a mixture of porcine and human N-acetyl renin substrates which differ in their four carboxyl-terminal residues resulting in a mass difference of about one Da. Both peptides had a free carboxy terminus.
Of the 74 laboratories that requested the samples, 26 (35%) returned data from 28 instruments. A majority of analyses were performed on MALDI-TOF instruments (64%) using the different modes available (linear, reflector, delayed extraction), 32% used electrospray coupled to quadrupole instruments, and one sample set was analyzed using an electrospray ion trap. All participants were able to report a mass for MS-1 and 50% could correctly identify the compound, while 19% were able to identify both MS-1 and MS-2. In most laboratories where the identification of the compounds was attempted, collision-associated dissociation (CAD) was used; several laboratories succeeded using enzymatic carboxyl-terminal ladder sequencing to obtain partial sequence information (3 to 7 residues). Ladder sequence analysis was performed mostly on MALDI-TOF instruments. All laboratories used database search routines to some extent and stated that this played a crucial role for positively identifying the compounds.
In general, a large variation in accuracy and resolution was seen among different laboratories and, in some cases, even within the same laboratory. It is difficult to compare the data from different participants in terms of their accuracy and quality due to a large variety of different instrumentation used, but the data suggest that improvement in methodology or procedures probably would have resulted in more comparable results in data sets within individual laboratories. In some data sets the resolution and accuracy obtained, while not as high as might be achieved with the latest instrumentation, still allowed identification of the peptide components. This suggests that perhaps the major contribution of the newer technology in achieving high-accuracy and high- resolution mass measurements is that it allows faster results with greater efficiency without resorting to adjunct procedures such as HPLC separation of components, ladder sequencing, or repetitive statistical methods.
Return to the The ABRF Home Page