This paper describes development of a direct selected reaction monitoring (SRM) mass spectrometric method for measuring Aβ42, Aβ40, and Aβ38. This technique avoids using antibodies to capture Aβ peptides and their possible bias due to affinity and epitope binding properties. The authors chose cerebrospinal fluid (CSF) as the calibrator matrix and a guanidine hydrochloride sample treatment protocol followed by a mixed-bed ion exchange “cleanup” step, as recently described (Lame et al., 2011). Keep in mind that the majority of proteomic mass spectrometry analyses have to use proteolytic digestion of the sample prior to the SRM mass spectrometry. However, since Aβ peptides have mass-to-charge ratios that are within the limits of mass spectrometers, the analysis can avoid the proteolytic digestions. Thus, we have a golden opportunity to once and for all measure Aβ in the CSF directly, free from matrix effects, and with adequate sensitivity, as demonstrated in this paper. And, as the authors note, there is a joint project among four labs that is well underway to develop both a...  Read more

This paper describes development of a direct selected reaction monitoring (SRM) mass spectrometric method for measuring Aβ42, Aβ40, and Aβ38. This technique avoids using antibodies to capture Aβ peptides and their possible bias due to affinity and epitope binding properties. The authors chose cerebrospinal fluid (CSF) as the calibrator matrix and a guanidine hydrochloride sample treatment protocol followed by a mixed-bed ion exchange “cleanup” step, as recently described (Lame et al., 2011). Keep in mind that the majority of proteomic mass spectrometry analyses have to use proteolytic digestion of the sample prior to the SRM mass spectrometry. However, since Aβ peptides have mass-to-charge ratios that are within the limits of mass spectrometers, the analysis can avoid the proteolytic digestions. Thus, we have a golden opportunity to once and for all measure Aβ in the CSF directly, free from matrix effects, and with adequate sensitivity, as demonstrated in this paper. And, as the authors note, there is a joint project among four labs that is well underway to develop both a reference method and reference material for Aβ42 measurements. We can enjoy the data presented in this paper, and even more to come from participants in that group.

This method improves on other CSF Aβ measures by its freedom from matrix effects, its ability to determine total Aβ42 as compared to the free form that immunoassays measure, and its accuracy-based approach. However, fully qualifying an analytical method for use in research studies and treatment trials requires lots of repetitions to ensure calibrator linearity, quality control reproducibility, and sufficient comparisons between samples from AD patients and various control groups. In addition, from the perspective of one whose lab does lots of mass spectrometry method development and implementation, there is a lot of room for further assessments of what columns work best and how to sustain stable sensitivity of the mass spectrometer. It usually takes thousands of injections of sample preparations to nail this down.

No question—this is a labor-intensive procedure. We have been working with our version of this type of approach for approximately one and a half years in our lab at UPenn. I am sure that the mass spectrometry companies will become engaged in this field if they see a market. Then they will put more resources into it, and with efforts from investigators in the field, that could lead to improvements in the efficiency of this type of methodology. As it stands now, based on my experience in both the research lab and the clinical lab arena, this will be primarily a research tool, useful for tracking Aβ42 and other Aβ peptides in treatment trials. Immunoassays are likely the mainstay methodology for the foreseeable future in this field as far as the clinical lab is concerned. That we don’t have candidate mass spectrometry methods for other important CSF biomarkers such as tau, α-synuclein, and TDP-43, etc., is all the more reason for the predominant use of immunoassays for the CSF AD biomarkers.

I agree with the authors that follow-up to this pilot study is needed. Larger numbers of study subjects, better calibrations, additional testing in patient cohorts, and inter-laboratory study (which appears well along in the planning stages), are all needed. This is a very demanding type of methodology that does require a high level of skilled personnel.

It is interesting to see, albeit on a very small number of study subjects, some improvement in the separation between AD patients and controls using the Aβ42/Aβ40 ratio. This will need to be confirmed by the authors and others, but if this can be replicated, it could provide somewhat better—more specific—AD detection. Also, and importantly, these pilot data do lead to the preliminary impression that the immunoassay (ELISA) provides comparable receiver operator characteristics and area under the curves for the AD versus normal controls, and if replicated that would provide important support for the clinical utility of Aβ measurement using currently available research immunoassays.

References:
Lame ME, Chambers EE, Blatnik M. Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry. Anal Biochem. 2011 Dec 15;419(2):133-9. Abstract

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