Mark Duncan (Presenter)
School of Medicine, University of Colorado
Bio: Mark Duncan is a professor of medicine at the University of Colorado School of Medicine. He has a long-standing interest in the application of mass spectrometry to clinical chemistry.
Authorship: Mark W. Duncan1, Stephen J. Hattan,2 Kenneth C. Parker,2 Jane Y. Yang,3 David A. Herold,3,4 and Marvin L. Vestal2
1 University of Colorado School of Medicine, 2 SimulTOF Systems,3Department of Pathology, University of California San Diego and 4 VA San Diego Healthcare System
MALDI-TOF MS, an established technique for analyzing nonvolatile molecules, has limited acceptance for clinical applications as a qualitative tool due to the widespread belief that MALDI-TOF is not quantitative. However, modern MALDI-TOF mass spectrometers generate reproducible data that can be used for accurate quantification of components in complex samples, such as whole blood and urine. Clinical MALDI applications require a very small volume of sample (e.g., 1 µL), and in some instances workup can be as simple as dilution. Other applications may require addition of an internal standard or purification and concentration of the analyte(s). Clinical assays illustrating each of these cases will be described.
Clinicians generally perceive mass spectrometry as being time intensive and costly. This perception is in part fueled by the dominance of LC-MS and LC-MS-MS in the products provided by major vendors. Recent FDA approval of MALDI-TOF MS methods for pathogen identification is beginning, however, to change this perception.
New MALDI-TOF instruments designed for routine clinical applications can be fully automated and require little or no expertise in MS on the part of the operator. The user prepares the samples according to a protocol established for the application, loads the sample plates into the instrument, and receives and interprets the results. The instrument parameters are determined from data provided along with the samples, and data acquisition, processing, and database searching are fully automated. No other interaction between the user and the instrument is required.
MALDI methods can be developed to analyze any bodily fluid containing an analyte of interest: e.g., blood and blood products, breast milk, cerebrospinal fluid, lymph fluid, saliva, urine, gastric and digestive fluid, tears, stool, semen, prostatic fluid, vaginal fluid, amniotic fluid, and interstitial fluids derived from tissue. Details of sampling and processing depend on the application, but the same MALDI-TOF instrument is common for all. The features of leading-edge MALDI instruments that make routine clinical application possible will be illustrated, and examples of successful MALDI-TOF MS assays will be presented.
For example, the determination of HbA1c, a glycated form of the β chain of hemoglobin (Hb), is important in the diagnosis and management of diabetes. Due to the high concentration of Hb in blood, all that is required for analysis by MALDI-TOF is an ~ 1 : 2000 dilution of whole blood. The analysis is both precise and accurate across the clinically relevant range INCLUDE THE RANGE with CV ≤ 3%. Results are reported as a percentage of HbA1c relative to the abundance of the unmodified β chain of Hb. (Because both species are present in the sample, no external standard or calibrant is required.)
The presence of albumin in the urine, or albuminuria, is typically as a symptom of kidney disease. MALDI-TOF MS can be used to detect and quantify urinary albumin following a ~ 1:10 dilution of a random (spot) urine sample. Because absolute quantification is required in this instance, either an internal standard must be added, or the approach of standard addition must be used. We have shown that the measurement urinary albumin is linear (R2 = 0.99) and quantitative from 5 – 500 mg/L of albumin. We also quantified urinary creatinine by this approach using deuterated creatinine-[2H3] as an internal standard. Measurements of urinary creatinine were made from 300 – 4000 mg/L with a CV < 10%.
Often, due to the complexity of the sample matrix or the low concentration of analyte(s) of interest, a workflow involving clean up and/or target enrichment is necessary. Mass spectrometric immunoassay (MSIA) is just one successful example. The MISA technique uses affinity purification for the specific capture and concentration of a targeted analyte prior MALDI-TOF MS detection and has been used to quantify a range of clinically relevant proteins and peptides. Generally, the affinity resin is housed in a pipet-tip and, post capture, the analyte is eluted directly onto the MALDI-TOF target. This workflow is easily scaled for high-throughput and a recent study on the analysis of insulin-like growth factor reports the processing of > 1000 samples in a single day. Similarly, a technique known as stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) involves cleavage of the intact polypeptides to peptide fragments prior to detection and quantification of specific peptides by MALDI-TOF MS. Examples of these approaches will also be presented.
In summary, MALDI-TOF is making inroads into routine clinical chemistry. Of special interest and relevance is the potential for MALDI to serve as a low cost, high throughput and precise analytical tool for quantitative analysis. The strengths and weaknesses of this approach will be presented and discussed.
References & Acknowledgements:
IP Royalty: no
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