Key points are not available for this paper at this time.
Currently, there are high degrees of both enthusiasm and controversy regarding the potential diagnostic application of matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry (1)(2)(3)(4)(5)(6)(7). This technique permits the simultaneous analysis of a large number of polypeptide components in biological fluids. Greatest sensitivity and resolution are achieved by MALDI TOF mass spectrometry in the mass range from ∼500 to 20 000 Da; this has led to the recognition that there is a highly complex mixture of peptide components in serum that circulate bound to larger proteins (8)(9). The complex patterns of peptide components are very information rich and may contain multiple biomarkers of diagnostic value (1)(2)(3)(4)(5)(6)(7)(8)(9). The present study examined whether different types of blood collection tubes add molecules to specimens that may appear as interfering or confounding peaks during MALDI TOF mass spectrometry. Commercially available blood collection tubes contain multiple components that may shed polymers in the molecular size range of interest. Silicones are commonly used as lubricants for stoppers or coatings for the internal surface of tubes. Polymeric surfactants such as polyvinylpyrrolidones or polyethylene glycols may be added to influence surface wetting. Tubes may contain either clot inhibitors or activators. Serum separator tubes contain polymeric gels with several constituents to adjust viscosity, density, and other physical properties. Rubber stoppers and the plastics comprising tube walls may shed polymeric components or plasticizers. Previous studies have reported effects of blood collection tubes on a variety of laboratory tests (10)(11)(12)(13)(14). These effects can arise from adsorption of serum or plasma components to the tube or separator gel, from the release of materials that interfere with specific test procedures, or from variable clotting of specimens. Although the potential effects of blood collection tubes need to be considered for any laboratory test, this may be particularly important for MALDI TOF mass spectrometry-based laboratory tests, which measure a broad spectrum of different components in a single analysis. We examined the shedding of components from tubes into aqueous solutions by adding 1 mL of phosphate-buffered saline (pH 7.2; KD Medical) to collection tubes and incubating the tubes at room temperature for ∼4 h with gentle rocking to allow contact with all surfaces of the tubes. This was considered to simulate typical contact times of blood specimens from collection to processing or analysis. Tubes tested included multiple types from two manufacturers, Becton Dickinson and Greiner Bio-One Vacuette North America (Table 1 ). After incubation in tubes, saline solutions were mixed with one-half volume of 6 mol/L guanidine hydrochloride containing 3 g/L trifluoroacetic acid. We extracted 30-μL aliquots by solid-phase extraction on Millipore C18 ZipTips® with a bed volume of 0.6 μL of reversed-phase packing. Tips were washed three times with 1 g/L trifluoroacetic acid before materials bound to the solid phase were eluted with 3 μL of 500 mL/L acetonitrile containing 1 g/L trifluoroacetic acid. Equal volumes (1 μL) of eluate and 50 mmol/L α-cyano-4-hydroxycinnamic acid or sinapinic acid matrix in 500 mL/L acetonitrile containing 1 g/L trifluoroacetic acid were mixed on a 384-position target plate and dried. Analyses were performed with a Bruker UltraFlex® MALDI TOF mass spectrometer in positive linear mode as described previously (15). Briefly, data were summed for 300 laser pulses collected from 10 positions. Measurements of mass/charge (m/z) were by external calibration. Calibrators, α-cyano-4-hydroxycinnamic acid, and sinapinic acid were purchased from Bruker Daltonics. Peaks observed by MALDI TOF mass spectrometry in extracts of blood collection tubes.1 Results were obtained with α-cyano-4-hydroxycinnamic acid as matrix. Peaks observed by MALDI TOF mass spectrometry in extracts of blood collection tubes.1 Results were obtained with α-cyano-4-hydroxycinnamic acid as matrix. Serum collection tubes, except for a no-additive glass tube, contributed components that yielded a complex series of peaks with a constant m/z interval in the m/z range from ∼1000 to 3000 (Fig. 1 and Table 1 ). Assuming that the primary components have a single positive charge, the series appeared as groups of five to six peaks differing by 14 Da and repeating every 111 Da. The 111-Da repeating unit was consistent with the monomer mass for polyvinylpyrrolidone, whereas the 14-Da unit may correspond to variation in the number of methylene groups (-CH2-) or methyl for hydrogen substitutions in the polymer. These were observed most clearly when we used the α-cyano-4-hydroxycinnamic acid matrix, which generally yielded high signal intensities for components in the m/z range 20. There were a few potential small peaks, but these could not be confidently distinguished from noise. By comparison, when we used the signal/noise ratio >20 cutoff, we identified 13 peaks in the spectrum in Fig. 1A and 33 in Fig. 1B . The spectrum for an extract from a research-use plasma separator tube designed to stabilize proteins for analysis (Fig. 1E ) contained several peaks with m/z of ∼1052, 1066, 6509, 6696, and 6880 (Fig. 1E ), but there was no series of peaks corresponding to a pattern for polymeric components as observed for the serum separator tubes in Fig. 1 , A and B. The signal/noise ratios for the peaks at m/z 1052 and 1066 were substantially >20; the identities of these components are unknown. The computer algorithm did not register measures of signal/noise ratio for the peaks at m/z 6500–7000 because of the greater widths of these peaks. These peaks were considered to possibly represent aprotinin or other similar protease inhibitors added to the tube. Purified bovine aprotinin (Sigma-Aldrich) yielded a single peak coinciding with the peak from the collection tube with m/z ∼6509 (not shown). Analysis of other types of tubes for plasma collection containing citrate or heparin as additives yielded spectra with no obvious peaks (not shown). Although heparin is a heterogeneous biopolymer with a molecular mass of ∼10 000, it has a strong negative charge that should lead to low detection in a positive ion mode. Heparin also is a highly polar oligosaccharide that might not be retained by the reversed-phase matrix used for sample preparation. When we used sinapinic acid as the sample matrix, which provides better detection of components with m/z >5000, extracts from blood collection tubes revealed no peaks that could be attributed to heparin (not shown). From the results described above, we conclude that most types of commonly used blood collection tubes for serum collection add polymeric components that can be detected by MALDI TOF mass spectrometry in the m/z range 1000–3000. These peaks potentially complicate and compromise the interpretation of the patterns of low-molecular-weight peptides. A variety of procedures have been used to fractionate serum components before mass spectrometric analysis (1)(2)(3)(4)(5)(6)(7)(8)(9). Whether the polymeric components from blood collection tubes will appear in the final mass spectra of these analyses is difficult to predict and will depend on multiple factors, such as interactions of polymers with plasma components, interactions with chromatographic phases, and relative ionization efficiency of polymeric components vs polypeptide components. Special research-use blood collection tubes designed to minimize peptide and protein breakdown in specimens may contain a variety of protease inhibitors. The particular type of tube tested in this study contained several components approximately the size of aprotinin. In view of the potential interference of components shed from blood collection tubes on the mass spectrometric analysis of low-molecular-weight serum polypeptides, researchers examining patterns of low-molecular-weight peptides and proteins for diagnostic purposes would be advised to take the following precautions: (a) The type of collection tube used for a diagnostic application should be standardized, as should the procedure for specimen processing and handling. Use of a diverse range of collection tubes and procedures may complicate the interpretation of mass spectrometric analysis of serum banks collected from multiple sites or over an extended period of time during which types of collection tubes have changed. (b) Collection tubes should be tested for interference in the analysis of interest. Examination of the components eluted from tubes into a saline solution, as performed in this study, offers a simple initial check for potential interfering components. Use of saline rather than a biological fluid such as serum has the major advantage of not requiring that researchers must decide whether observed peaks represent peaks derived from the tubes or from the specimens. However, there is the caveat that this approach provides an imperfect survey of interferences with complex biological specimens. It could underestimate some potential interfering materials that would be extracted more efficiently by protein-containing solutions such as blood. This approach also may identify some potential interferents that will not be significant in a particular analysis because they will be removed by preanalytical processing steps or are present at much lower concentrations than endogenous components. Finally, some tube effects might result from extraction of specific components or interactions with the specimen, such as activation of proteases, and these types of effects would not be identified by the present type of study. Some types of tubes designed to reduce protein degradation may contain aprotinin or other protease inhibitors that will appear as peaks in mass spectra. These components are potential interferents if they are not recognized as exogenous additives to a specimen. However, addition of exogenous protein components may not always be an undesirable characteristic of collection tubes if the added components do not overlap or mask endogenous components of interest. As well as controlling proteolysis, addition of exogenous protein components might serve useful roles such as internal standardization for recovery or mass calibration. Collection of plasma may offer an attractive option to serum collection in that it appears to add fewer extraneous polymers from collection tubes, and greater consistency in processing is expected because there is no variation in clotting kinetics or endpoints, as there is in generation of serum. In addition, plasma would not contain the additional activation peptides from the clotting process or small peptides released from platelets, and chelation of metals with additives such as EDTA should reduce the peptidolytic activity of specimens. There is a need for further systematic evaluation of the effects of collection materials and processing procedures to apply MALDI TOF mass spectrometry reliably for diagnostic applications. Previous studies on the effects of blood collection materials and processing procedures on many other types of laboratory analyses (10)(11)(12)(13)(14) suggest that optimization and standardization of collection procedures will be an important element in reliable analysis of serum or plasma proteins by mass spectrometry for diagnostic purposes.
Drake et al. (Wed,) studied this question.