TY - JOUR
AU - Hess,, Rüdiger
AB - Abstract Background: The Val34Leu mutation in the activation peptide of factor XIII (FXIIIA) correlates with a lower incidence of myocardial infarction and ischemic stroke but an increased risk for hemorrhagic stroke. We describe mass spectrometric detection of the activation peptide variants in human serum. Methods: We used differential peptide display (DPD) to compare comprehensive peptide maps from pairs of serum samples from healthy volunteers. Peptides were separated by liquid chromatography, and fractions were subjected to mass spectrometry. Mass spectra of all fractions were combined, giving a peptide map representing a two-dimensional display of peptide masses. After comparison of peptide mass maps, peptides that differentiated FXIIIA phenotypes were identified by mass spectrometry. Results: Val34Leu polymorphisms of the activation peptide of FXIIIA were identified in 20 serum samples from 10 volunteers by DPD, and their sequences were confirmed by nanoelectrospray-ionization quadrupole time-of-flight mass spectrometry. Analysis of three (V34V, V34L, and L34L) phenotypes was confirmed by allele-specific genotypic analysis in all (n = 10) volunteers. Conclusion: DPD provides a simple and easy-to-use phenotype assay with advantages over PCR-based assays in being faster and directly analyzing the compound of interest. In the final step of the clotting cascade, coagulation factor XIII A chain (FXIIIA) 1 is activated by thrombin-catalyzed cleavage of its activation peptide. Active FXIIIA generates intermolecular amide bonds between lysine and glutamine residues, leading to covalent cross-linking of fibrin strands and conversion of soluble fibrin molecules into a stable insoluble clot. The involvement of FXIII in cardio- and cerebrovascular diseases has been investigated recently, and the presence of a Val34Leu mutation is known to correlate with a lower incidence of myocardial infarction and ischemic stroke but an increased risk for hemorrhagic stroke (1)(2)(3)(4)(5). This mutation is localized in the direct vicinity of the cleavage site and is thought to influence the activation process of this protein (1). To date, allelic genotypes have been identified by PCR with restriction fragment length polymorphism analysis (4) or by allele-specific PCR (6). Although the reliability of these molecular biology techniques has been demonstrated, some restrictions remain because of the indirect analysis of a highly amplified descendant of the real target. Additionally, careful handling of the sample material is essential to reduce the risk of contamination in diagnostic PCR. Finally, only samples containing DNA/RNA can be used for such analyses. Differential peptide display (DPD™; BioVisioN) (7)(8)(9) is a technique that generates comprehensive peptide maps of ∼3000 peptides covering a mass range of 950–15 000 Da from a biological sample. The sample is separated by reversed-phase HPLC (RP-HPLC; Fig. 1A ), and the peptides eluting from the HPLC column are collected in 96 fractions. Each fraction is subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; Fig. 1B ), and the mass spectra of all 96 fractions are combined, producing a two-dimensional display of peptide masses in which the abscissa displays the mass-to-charge ratio, the ordinate is determined by the retention time on the RP-HPLC column, and the signal intensity is depicted by color saturation (Fig. 1C ). This display method allows the processing and analysis of large amounts of data. Peptide maps of individual samples can be superimposed, facilitating the detection of differences in the resulting subtractive peptide maps (Fig. 1D ). For the identification of peptides, peaks from individual HPLC fractions can be subjected to nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nESI-qTOF-MS) sequencing, which gives peptide fragment spectra (Fig. 1E ). These spectra serve to identify the corresponding peptide sequences by remote database searching. Figure 1. Open in new tabDownload slide DPD analysis of serum samples. Protein-depleted serum samples were separated by RP-HPLC into 96 fractions (A). Each fraction was analyzed by MALDI-TOF-MS, and the resulting mass spectrum (B, top) was transformed into a one-dimensional virtual gel with a bar pattern (B, bottom). All 96 of these single-fraction patterns were combined, giving a two-dimensional peptide map (C). For each sample, mean peptide master maps derived from several individual maps of the same sample were created. Two such mean master maps derived from different donors are displayed in different colors (red, homozygous VV; blue, homozygous LL) and superimposed. This differential peptide map indicates differences in peptide abundance by differences in the amounts of the respective colors (D). The Roman and Arabic numerals indicate differentially expressed peptides. To determine the amino acid sequences of particular peptides, the corresponding RP-HPLC fractions were subjected to nESI-qTOF-MS sequencing (E). Figure 1. Open in new tabDownload slide DPD analysis of serum samples. Protein-depleted serum samples were separated by RP-HPLC into 96 fractions (A). Each fraction was analyzed by MALDI-TOF-MS, and the resulting mass spectrum (B, top) was transformed into a one-dimensional virtual gel with a bar pattern (B, bottom). All 96 of these single-fraction patterns were combined, giving a two-dimensional peptide map (C). For each sample, mean peptide master maps derived from several individual maps of the same sample were created. Two such mean master maps derived from different donors are displayed in different colors (red, homozygous VV; blue, homozygous LL) and superimposed. This differential peptide map indicates differences in peptide abundance by differences in the amounts of the respective colors (D). The Roman and Arabic numerals indicate differentially expressed peptides. To determine the amino acid sequences of particular peptides, the corresponding RP-HPLC fractions were subjected to nESI-qTOF-MS sequencing (E). In the present study, we identified the activation peptide (amino acids 1–37) cleaved from the NH2 terminus of FXIIIA and several truncated variants in serum samples by DPD; the exchange of valine to leucine was detected by a mass difference of 14 Da and a chromatographic shift of four fractions. The peptide sequences were confirmed by nESI-qTOF-MS. We also identified this polymorphism by direct MALDI-TOF-MS measurement in unfractionated serum samples. Material and Methods blood samples After receiving written informed consent, we enrolled 10 healthy adult men in this study; the local Ethics Committee at the Hanover Medical School approved the protocol. Blood samples were collected from the cubital vein into blood collection tubes (9-mL S-Monovette with clot activator; Sarstedt) at one time point. The samples were then made anonymous. After the blood had coagulated (∼1 h), the tube was centrifuged at 2000g for 10 min at 4 °C. The serum was separated from the clot and stored at −80 °C until analysis. sample preparation Before analysis, the protein load in the serum samples is reduced by precipitation with trichloroacetic acid. Serum (0.5 mL) was added to 1 mL of ice-cold distilled water. For protein precipitation, 0.5 mL of a solution containing 200 g/L trichloroacetic acid was added, and the tubes were vigorously mixed for 30 s. After incubating for 30 min on ice, samples were centrifuged at 18 000g for 30 min at 4 °C. The supernatants were transferred to sample vials. allelic genotyping Genotyping for the FXIII V34L polymorphism was performed at a professional laboratory for genetic analyses (IMBA, Weinitzen, Austria) by an allele-specific amplification assay (6). dpd analysis Serum extracts were loaded on a RP-HPLC column. The peptides were eluted by an acetonitrile gradient (4–40%), and 96 fractions were collected. The lyophilized fractions were resuspended in a mixture of α-cyano-4-hydroxycinnamic acid (matrix) and 6-desoxy-l-galactose (comatrix) in acetonitrile containing 1.5 g/L trifluoroacetic acid (TFA) and subjected to MALDI-TOF-MS (10) in a Voyager DE STR (Applied Biosystems). Data were analyzed, including peak recognition and visualization, with the in-house-developed software package Spectromania™ (BioVisioN). MS signals were quantified after baseline correction by integration of absolute signal intensities in 1-Da bins. The Spectromania software package also transformed the mass spectrum of each fraction to a virtual one-dimensional gel; the molecular weight of each peptide is indicated by its position within the virtual gel lane, whereas the MALDI signal intensity for each peptide is indicated by the color intensity of the corresponding bar. The converted mass spectra of all 96 fractions were combined to give a two-dimensional display of peptide masses, termed a peptide map (Fig. 2 ). Peptide maps of individual samples were superimposed, generating mean peptide maps. Differentially expressed peptides were detected by calculation of subtractive peptide maps. Figure 2. Open in new tabDownload slide Virtual two-dimensional DPD map. The DPD map (equivalent to 300 μL of serum) was generated from a serum sample from a heterozygous donor. Each lane represents a HPLC fraction and each band a peptide. Peptides derived from the NH2 terminus of FXIIIA are indicated as V (wild type) or L (mutant). The peptide masses are given in Table 1 . Figure 2. Open in new tabDownload slide Virtual two-dimensional DPD map. The DPD map (equivalent to 300 μL of serum) was generated from a serum sample from a heterozygous donor. Each lane represents a HPLC fraction and each band a peptide. Peptides derived from the NH2 terminus of FXIIIA are indicated as V (wild type) or L (mutant). The peptide masses are given in Table 1 . identification by nESI-qTOF-tandem MS Selected peptides were identified by sequencing with a nESI-qTOF tandem mass spectrometer (QSTAR pulsar; Sciex) with subsequent protein database searching. The resulting peptide fragment spectra were produced in the product-ion scan mode (spray voltage, 950 V; collision energy, 28 eV). A total of 200 scans per sample were accumulated. Data processing before database searching included charge state deconvolution (Bayesian reconstruct tool of the BioAnalyst program package; Sciex) and deisotoping (customized Analyst QS macro; Sciex). The resulting spectra were saved in MASCOT (Matrix Science) generic file format and submitted to the MASCOT search engine. Cascading searches including several posttranslational modifications in SwissProt (Ver. 41; www.expasy.ch) and MSDB (Ver. 030212; EBI) were performed by the MASCOT DEAMON client software (Ver. 1.9; Matrix Science). maldi-tof-ms analysis of unfractionated serum samples For sample preparation before MALDI-TOF-MS, ZipTip devices (Millipore Corporation) were equilibrated with 10 μL of 1 g/L TFA. Deproteinized serum (5 μL) was aspirated into the device, and the device was washed three times with 1 g/L TFA. Peptides were eluted directly onto the MALDI target by use of a mixture of α-cyano-4-hydroxycinnamic acid (matrix) and 6-desoxy-l-galactose (comatrix) in acetonitrile (580 mL/L) containing 1 g/L TFA. MALDI-TOF-MS analysis was performed with a Voyager STR mass spectrometer (Applied Biosystems). surface-enhanced laser desorption/ionization ms analysis Surface-enhanced laser desorption/ionization (SELDI)-TOF-MS (11)(12) was performed with WCX chips (weak cation-exchange capacities), SAX chips (anion-exchange capacity), and immobilized metal-affinity capture (IMAC) chips (metal-chelating capacity). Because of the low detection potential of hydrophobic chips and the high costs of the expendable SELDI targets, H4 chips were not used in this study. Approximately 5 μg of protein was diluted 1:10 in the respective binding buffers and applied to the chip by a bioprocessor (Ciphergen Biosystems Inc.). After 30 min, samples were removed, and the chips were washed with the respective washing buffers followed by two quick washes with water. Two 0.7-μL portions of freshly prepared matrix were then added to each spot. Cyano-4-hydroxycinnamic acid (10 g/L) as matrix and 6-desoxy-galactose (10 g/L) as comatrix in 500 mL/L acetonitrile containing 5 g/L TFA were used. WCX chips were preactivated by the addition of 10 mmol/L HCl to the spots followed by three washes with H2O. As binding and washing buffer, 20 mmol/L ammonium acetate, pH 4.0, was used. SAX chips were activated by the addition of 5 μL of 50 mmol/L HEPES buffer, pH 7.5, containing 1 mL/L Triton X-100, which was also used as binding and washing buffer. IMAC chips were pretreated with water and loaded with Ni2+ by use of 20 μL of 100 mmol/L NiSO4. Excess Ni2+ was removed by rinsing with water. As binding and washing buffer, 50 mmol/L Tris, pH 7.5, containing 300 mmol/L NaCl and 0.2 mL/L Triton X-100 was used. Additionally, all samples were analyzed with a gold chip. Extracts were mixed with matrix (see above), applied to the chip, dried, and analyzed in the Ciphergen mass spectrometer. chemicals All laboratory chemicals were obtained from Merck AG if not otherwise indicated. All laboratory chemicals for SELDI-MS analysis were obtained from Sigma-Aldrich GmbH. Results detection and identification of the val34leu polymorphism by dpd To identify the Val34Leu polymorphism in the activation peptide of FXIIIA, blood samples from 10 healthy volunteers were subjected to DPD analysis and individual peptide maps were generated. Among other differences in these maps, two mass peaks with mass-to-charge ratios of 3950 and 3964 allowed separation of the individuals into three subgroups. We used accumulated data in our peptide tracking application (PeTrA) database, in which ∼2500 peptide sequences are stored, to predict the identities of the peptides. The mass of 3950 Da corresponded to the deduced mass of the FXIIIA activation peptide with valine at position 34; the mass peak at 3964 Da corresponded to a similar peptide with leucine at position 34. In addition, signals with m/z ratios of 3349/3363, 2759/2773, and 2602/2616 with a similar pattern corresponding to the above-mentioned subgroups were detected. nESI-qTOF-MS analysis confirmed the identities of the detected mass peaks. The peak with a mass-to-charge ratio of 3950 Da belonged to the activation peptide of FXIIIA (amino acids 1–37), whereas the peak with a mass-to-charge ratio of 3964 Da also belonged to the activation peptide of FXIII containing leucine at position 34 (Table 1 ). The other differentially detected signals belonged to truncated forms of the activation peptide: 3350/3364 belonged to a peptide comprising amino acids 6–37, 2759/2773 to a peptide comprising amino acids 12–37, and 2602/2616 to a peptide comprising amino acids 13–37 (Table 1 ). In each case, the 14-Da higher mass corresponded to the Leu-34 variant. These peptides might be generated by action of proteases such as thrombin and plasma kallikrein (Table 1 ). Our observation with 10 volunteers shows that in all cases in which the VV phenotype was detected, no peptide signals were detected at the positions of the LL phenotype and vice versa (Fig. 3 ). This was true for all four peptides that were derived from the activation peptide. This means that, in all cases of VV phenotype, only peaks with m/z values of 3950, 3349, 2759, and 2602 were detected from the respective pairs. For the LL phenotype, only peaks with m/z values of 3964, 3363, 2773, and 2616 were detected from the respective pairs. Table 1. Activation peptides and fragments.1 Index . AA2 sequence . m/z observed . Peptide generated through putative cleavage by . I SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3950 Thrombin (EC 3.4.21.5) II TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3349 Plasma kallikrein (EC 3.4.21.34) III RAVPPNNSN AAEDDLPTVE LQGVVPR 2759 Plasma kallikrein (EC 3.4.21.34) IV AVPPNNSN AAEDDLPTVE LQGVVPR 2602 Plasma kallikrein (EC 3.4.21.34) 1 SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3964 Thrombin (EC 3.4.21.5) 2 TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3363 Plasma kallikrein (EC 3.4.21.34) 3 RAVPPNNSN AAEDDLPTVE LQGVVPR 2773 Plasma kallikrein (EC 3.4.21.34) 4 AVPPNNSN AAEDDLPTVE LQGLVPR 2616 Plasma kallikrein (EC 3.4.21.34) Index . AA2 sequence . m/z observed . Peptide generated through putative cleavage by . I SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3950 Thrombin (EC 3.4.21.5) II TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3349 Plasma kallikrein (EC 3.4.21.34) III RAVPPNNSN AAEDDLPTVE LQGVVPR 2759 Plasma kallikrein (EC 3.4.21.34) IV AVPPNNSN AAEDDLPTVE LQGVVPR 2602 Plasma kallikrein (EC 3.4.21.34) 1 SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3964 Thrombin (EC 3.4.21.5) 2 TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3363 Plasma kallikrein (EC 3.4.21.34) 3 RAVPPNNSN AAEDDLPTVE LQGVVPR 2773 Plasma kallikrein (EC 3.4.21.34) 4 AVPPNNSN AAEDDLPTVE LQGLVPR 2616 Plasma kallikrein (EC 3.4.21.34) 1 Listed are the peptides that were detected and sequenced. The index refers to Fig. 1 . The detected m/z ratio is shown. The fragments are putatively generated through cleavage by the stated enzymes. 2 AA, amino acid. Open in new tab Table 1. Activation peptides and fragments.1 Index . AA2 sequence . m/z observed . Peptide generated through putative cleavage by . I SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3950 Thrombin (EC 3.4.21.5) II TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3349 Plasma kallikrein (EC 3.4.21.34) III RAVPPNNSN AAEDDLPTVE LQGVVPR 2759 Plasma kallikrein (EC 3.4.21.34) IV AVPPNNSN AAEDDLPTVE LQGVVPR 2602 Plasma kallikrein (EC 3.4.21.34) 1 SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3964 Thrombin (EC 3.4.21.5) 2 TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3363 Plasma kallikrein (EC 3.4.21.34) 3 RAVPPNNSN AAEDDLPTVE LQGVVPR 2773 Plasma kallikrein (EC 3.4.21.34) 4 AVPPNNSN AAEDDLPTVE LQGLVPR 2616 Plasma kallikrein (EC 3.4.21.34) Index . AA2 sequence . m/z observed . Peptide generated through putative cleavage by . I SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3950 Thrombin (EC 3.4.21.5) II TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3349 Plasma kallikrein (EC 3.4.21.34) III RAVPPNNSN AAEDDLPTVE LQGVVPR 2759 Plasma kallikrein (EC 3.4.21.34) IV AVPPNNSN AAEDDLPTVE LQGVVPR 2602 Plasma kallikrein (EC 3.4.21.34) 1 SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3964 Thrombin (EC 3.4.21.5) 2 TAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPR 3363 Plasma kallikrein (EC 3.4.21.34) 3 RAVPPNNSN AAEDDLPTVE LQGVVPR 2773 Plasma kallikrein (EC 3.4.21.34) 4 AVPPNNSN AAEDDLPTVE LQGLVPR 2616 Plasma kallikrein (EC 3.4.21.34) 1 Listed are the peptides that were detected and sequenced. The index refers to Fig. 1 . The detected m/z ratio is shown. The fragments are putatively generated through cleavage by the stated enzymes. 2 AA, amino acid. Open in new tab Figure 3. Open in new tabDownload slide MS determination of allelic phenotype. Deproteinized serum samples from 10 donors (P1–P10) were analyzed by MS. Heterozygous (VL) and homozygous (VV and LL) donors are indicated. The arrow indicates the mass spectrometric shift of 14 Da (methyl residue) between valine and lysine. Figure 3. Open in new tabDownload slide MS determination of allelic phenotype. Deproteinized serum samples from 10 donors (P1–P10) were analyzed by MS. Heterozygous (VL) and homozygous (VV and LL) donors are indicated. The arrow indicates the mass spectrometric shift of 14 Da (methyl residue) between valine and lysine. In Caucasian individuals, ∼53% are homozygous for valine (VV), 8% are homozygous for leucine (LL), and ∼39% are heterozygous (VL). In the group we investigated, a similar distribution was determined. As expected, the two heterozygous individuals had lower peak intensities, which indicates lower concentrations of the activation peptide of both phenotypes in the blood of these individuals. To confirm our results, all 10 blood samples were analyzed by allele-specific genotyping for the FXIIIA Val34Leu polymorphism. The results obtained validated the results obtained by DPD analysis. detection of the val34leu polymorphism of fxiiia by maldi-tof-ms and seldi-tof-ms The Val34Leu polymorphism was also detectable in serum samples by a rapid procedure with solid-phase extraction before subsequent MALDI-TOF-MS analysis. With this approach, peptide masses of 3950 and 3964 Da were identified, which corresponded to the Val-34 and Leu-34 variants of the FXIIIA activation peptide, respectively. In addition, masses of 3349/3363 Da and 2759/2773 Da were detected, which corresponded to the truncated forms of the activation peptide that had also been observed by DPD. The polymorphism of FXIIIA identified by DPD was also observable in the spectra produced by the Ciphergen mass spectrometer. Protein-depleted serum samples from four selected individuals were applied to SELDI chips. Peptides derived from FXIIIA that were identified by DPD were not observed by SELDI-MS on chips with cation-exchange (WCX chip), anion-exchange (SAX chip), or metal-chelating (IMAC chip) surfaces. On the standard gold surface chip, peaks with m/z ratios of 3954 and 3967 were readily detected, which corresponded to the DPD-identified activation peptides (amino acids 1–37). The truncated forms of the activation peptide were not detected by the SELDI chromatographic or standard gold chips. Discussion We describe here the detection and identification of the Val34Leu polymorphism of FXIIIA by DPD. Peptide profiles of serum samples were constructed, and both variants of the activation peptide of FXIIIA were detected and subsequently identified by tandem MS sequencing (Fig. 4 ). No other peptides were detected or sequenced at the respective positions (chromatographic elution plus mass-to-charge ratio). We believe that it is very unlikely that isobaric peptides derived from other proteins led to identical patterns because this pattern occurred in four distinct molecular forms and at a strongly pronounced intensity corresponding to the high concentration of FXIIIA-derived fragments. Figure 4. Open in new tabDownload slide Tandem MS sequencing of the FXIIIA-derived peptides. Deconvoluted ESI-tandem MS spectra (top) of the V (A) and L (B) variants are shown. (C and D), relevant regions of the spectra (zoom) in A and B, respectively. On the basis of the difference between ions b33 and b34, the two variants can be unambiguously distinguished. The amino acids that differ in the variants are indicated in bold. Figure 4. Open in new tabDownload slide Tandem MS sequencing of the FXIIIA-derived peptides. Deconvoluted ESI-tandem MS spectra (top) of the V (A) and L (B) variants are shown. (C and D), relevant regions of the spectra (zoom) in A and B, respectively. On the basis of the difference between ions b33 and b34, the two variants can be unambiguously distinguished. The amino acids that differ in the variants are indicated in bold. In addition, a direct MALDI-TOF-MS approach accurately detected this polymorphism, enabling fast and simple routine diagnostics. After solid-phase extraction of peptides from serum samples, the activation peptide of FXIIIA and degradation products of this peptide were easily detected. MALDI-MS instrumentation has matured over recent years, and fast and reliable analysis is possible. This procedure presented here is well suited for the determination of the identified polymorphism in larger cohorts and in routine diagnostics. The identification is facilitated by the high serum concentration of FXIIIA (30 mg/L of serum) (13)(14). This example shows a possible use for mass spectrometric analyses in routine laboratories. The sensitive DPD method allows the detection of biologically relevant peptides and their identification. SELDI-TOF-MS or MALDI-TOF-MS measurement can then be optimized for detection of this specific compound. This is achieved by variation of chip surfaces (e.g., cation- or anion-exchange surfaces), by use of antibodies immobilized on surfaces, or as described here, by use of simple gold chips or by use of solid-phase extraction before MALDI-TOF-MS. These approaches are easily established in clinical laboratories and are user friendly, allowing the analysis of large numbers of samples in a short period of time. The Val34Leu polymorphism is directly detected by the described mass spectrometric techniques. No time-consuming amplification step is necessary, and the risk of contamination leading to false-positive results is relatively low compared with molecular analysis tools such as allele-specific PCR and PCR with restriction fragment length polymorphism analysis, for which a separate laboratory for sample preparation is also needed. The described method can also be used in retrospective studies in which the phenotype can be determined in samples from patients with known follow-up data for whom no DNA was available. This approach can also be used when ethical aspects prohibit the use of DNA. We thank Michael Schrader, Norbert Lamping, and Peter Schulz-Knappe for fruitful discussions during preparation of this manuscript and for the suggestion to combine ZipTip extraction with MALDI-MS analysis to straightforwardly detect FXIII fragments in serum. We thank Daniela Müller, Kerstin Kupke, and Kirsten Minkhart for their skillful technical assistance. 1 " Nonstandard abbreviations: FXIIIA, coagulation factor XIII A chain; DPD, differential peptide display; RP, reversed-phase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; nESI-qTOF-MS, nanoelectrospray ionization quadrupole time-of-flight mass spectrometry; TFA, trifluoroacetic acid; SELDI, surface-enhanced laser desorption/ionization; and IMAC, immobilized metal-affinity capture. References 1 Undas A, Sydor WJ, Brummel K, Musial J, Mann KG, Szczeklik A. Aspirin alters the cardioprotective effects of the factor XIII Val34Leu polymorphism. Circulation 2003 ; 107 : 17 -20. Crossref Search ADS PubMed 2 Incorvaia C, Costagliola C, Parmeggiani F, Gemmati D, Scapoli GL, Sebastiani A. Recurrent episodes of spontaneous subconjunctival hemorrhage in patients with factor XIII Val34Leu mutation. Am J Ophthalmol 2002 ; 134 : 927 -929. 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TI - Mass Spectrometric Phenotyping of Val34Leu Polymorphism of Blood Coagulation Factor XIII by Differential Peptide Display
JF - Clinical Chemistry
DO - 10.1373/clinchem.2003.028209
DA - 2004-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mass-spectrometric-phenotyping-of-val34leu-polymorphism-of-blood-vf6ldPI4Et
SP - 545
VL - 50
IS - 3
DP - DeepDyve
ER -