Stephen Hattan (Presenter)
SimulTof Systems
Bio: Since joining SimulTOF Systems in 2006, my research has focused on innovative ways for improving the interface between sample preparation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Prior to SimulTOF Systems, I spent 10 years working in the field of discovery proteomics. My roles centered on the development of innovative technologies and methodologies for carrying out large scale proteomic investigations by mass spectrometry.
Authorship: Stephen J. Hattan (1), Kenneth C. Parker (1), Marvin L. Vestal (1), Jang Y. Yang (2), David A. Herold (2,3), Mark W.Duncan (4)
(1) SimulTOF Systems, 60 Union Avenue, Suite 1-R, Sudbury, MA 01776 (2) Department of Pathology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-9113 (3) VAMC-San Diego,
| Short Abstract “Albuminuria” is elevated levels of serum albumin (SA) in urine (U) and can indicate kidney malfunction / disease. We demonstrated a novel means for quantifying albumin directly or in comparison to creatinine (C) in urine by MALDI-TOF mass spectrometry. Standard addition of albumin and deuterated creatinine (d3) into control urine produced a linear and quantitative response (R2 = 0.99 and 0.98) used to quantify both in patient samples across the relevant ranges of 5 – 500 mg/L C (SA/U) and 300 – 4000 mg/L (C/U) with CV < 10%. This MS-based method represents a simple, fast, attractive alternative to currently clinical methods. |
Long Abstract
Introduction:
Chronic Kidney Disease (CKD) or kidney malfunction as a complication of another systematic disorder (diabetes, high blood pressure etc.) can cause decline in blood filtration capability resulting in the passage of a blood-born proteins through the kidneys with expulsion in urine (U)(1-3). Clinical analyses for blood proteins in urine are performed to assess proper kidney function and routine analyses are often performed to monitor a diagnosed disorder (4,5). Serum Albumin (SA), the most abundant blood serum protein, is a common target in these clinical assays. The detection of elevated levels of SA in urine is termed “Albuminuria” and the amount detected can indicate the acute severity of kidney condition (6). Because of normal variability in urine content and volume within an individual, and between individuals, as a result of hydration, diet, physical active and circumstance, etc., multiple SA/U measurements are often made to span a 24 h period. Additionally, SA/U measurements are often made and compared to creatitine (C) levels within the same urine sample (C/U) and reported as a ratio. Creatinine, a metabolite of muscle, is normally expelled at a relatively constant rate and its measure is used as normalization factor for the dilution (water content) of a given urine sample (7,8).
Results & Discussion:
Demonstrated here-in is a method for quantifying SA directly from diluted urine samples using linear-mode MALDI-TOF mass spectrometry. Standard addition curve (data point =6) of SA added to control urine is used to produce a linear calibration (R2 =0.98- 0.99) for the quantitation of unknowns across the clinically relevant range of 5 – 500 mg/L (SA/U). All data points are collected in technical replication of 6x with average intra-sample CV for all data points = 6.3% (Figure 1). The method is simple and fast requiring 10x dilution urine sample in MALDI matrix and an MS analysis time of ~ 30s / sample. Currently, most clinically assays of SA/U are based on an immune-affinity type platform (9). These assays rely on the specificity of the antibody – antigen interaction are often followed by secondary reaction for detection. Immune-affinity assays can suffer from nonspecific binding of both primary and secondary antibodies and from cross-reactivity with non-target compounds of similar structure. Also, the signals used for quantitation are often light- based interactions or properties with the based solution (absorption, emission, light scatter, turbidity) and not a direct measurement of the analyte in question. MALDI-TOF analysis offers direct measurement of intact albumin and produces spectra containing multiply charged species that provide build-in corroborative analyte measurement. An internal standard (lysozyme) added to the MALDI matrix is used to normalize spectra across analyses and albumin quantitation is made directly from native albumin peak integration.
In addition to SA, MALDI-TOF analysis of urine can be used for quantitation of creatinine. Because of the inherent difference in molecular mass and typical urine concentration levels between of the two species, the analyses are currently performed as two separate MS scans. Deuterated creatinine standard (d3) is differently spiked into urine and integrated area of the standard response is used to create a calibration curve for quantitation of the native creatinine. As with SA/U measurement, single point measurements of C/U fluctuate as a result of hydration status at the time of urine collection (as well as factors of body mass, gender, age etc); regardless, the accurate measure of C/U in conjunction with SA/U measurements are extremely useful for assessing proper kidney function.
Methods:
Sample preparation:
Urine sample diluted 1:10 in MALDI matrix (10mg/mL HCCA, 75% ACN, 0.1 % TFA) Human Serum Albumin, Sigma Aldrich (St. Louis, MO), Creatinine-d3, Santa Cruz Biotech. (Dallas, TX).
Data collection and processing:
Data collected on SimulTOF 100 (SimulTOF Systems, Sudbury, MA) MALDI-TOF mass spectrometer in linear mode using positive-ion polarization. Individual spectra are the average of 100 laser shots collected over a 5000 – 35,000 Dalton mass range using an acceleration voltage 20 kV, focus mass 15000, laser pulse frequency 1 khz,laser pulse energy 9uj, scan rate 1mm/min at 100um raster to cover each sample position. Post acquisition, spectra passing a 20mV minimum intensity threshold are averaged together to create a single “spot-averaged” spectrum for each sample position. These spot-averaged spectra were calibrated using averaged mass value of the M+1 and M+2 ions for the internal standard added to matrix (Cytochrome C or Lysozyme) and all spectra are normalized to the M+1 ion of the internal standard. Calibration curves for SA in urine are constructed from the integration of SA signal and plotted as a function of concentration. For creatinine measurements, spectra are calibrated using known matrix ion species and the creatinine-d3 internal standard. Calibration curves for native creatinine quantitation are constructed by plotting the integrated area of C-d3 peak as a function of concentration.
Conclusion:
Demonstrated here-in is a method for quantifying albumin and creatinine directly from diluted urine samples using linear-mode MALDI-TOF mass spectrometry. Method development is discussed and its application for the quantitation of clinical samples is demonstrated. We believe this technology represents a simple, fast, attractive alternative to currently practiced clinical methods.
References & Acknowledgements:
1: de Jong PE, Gansevoort RT. Albuminuria in non-primary renal disease: risk
marker rather than risk factor. Nephrol Dial Transplant. 2010 Mar;25(3):656-8.
2: Mischak H, Rossing P. Proteomic biomarkers in diabetic nephropathy--reality
or future promise? Nephrol Dial Transplant. 2010 Sep;25(9):2843-5.
3: Jain S, Rajput A, Kumar Y, Uppuluri N, Arvind AS, Tatu U. Proteomic analysis
of urinary protein markers for accurate prediction of diabetic kidney disorder. J
Assoc Physicians India. 2005 Jun;53:513-20.
4: Chiarelli F, Verrotti A, Mohn A, Morgese G. The importance of microalbuminuria
as an indicator of incipient diabetic nephropathy: therapeutic implications. Ann
Med. 1997 Oct;29(5):439-45.
5: Mogensen CE, Damsgaard EM, Frøland A, Nielsen S, de Fine Olivarius N, Schmitz
A. Microalbuminuria in non-insulin-dependent diabetes. Clin Nephrol. 1992;38
Suppl 1:S28-39.
6: Sampaio E, Delfino VD. Assessing albuminuria in spot morning samples from
diabetic patients. Arq Bras Endocrinol Metabol. 2008 Dec;52(9):1482-8.
7: Thongboonkerd V. Practical Points in Urinary Proteomics. J Proteome Research
Nephrol. 2007;6:3881-3890.
8: Nagaraj N, Mann M, Quantitative Analysis of the Intra- and Inter-Individual Variability
of the Normal Urinary Proteome.J Proteome Research
Nephrol. 2011;10:637-645.
9: Koivunen ME, Krogsrud RL. Principles of Immunochemical Techniques Used in Clinical Laboratories
Labmedicine 2006; 37: 490-497.
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