The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS.

OBJECTIVES: Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. METHODS: VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. RESULTS: Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. CONCLUSIONS: VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum.

OBJECTIVES: Matrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy. METHODS: Serum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only). RESULTS: Certified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003). TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS. CONCLUSIONS: The analyte concentrations' changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.

Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins.

Intact protein analysis by liquid chromatography-mass spectrometry (LC-MS) is now possible due to the improved capabilities of mass spectrometers yielding greater resolution, mass accuracy, and extended mass ranges. Concurrent measurement of post-translational modifications (PTMs) during LC-MS of intact proteins is advantageous while monitoring critical proteoform status, such as for clinical samples or during production of reference materials. However, difficulties exist for PTM identification when the protein is large or contains multiple modification sites. In this work, analyses of low-abundance proteoforms of proteins of clinical or therapeutic interest, including C-reactive protein, vitamin D-binding protein, transferrin, and immunoglobulin G (NISTmAb), were performed on an Orbitrap Elite mass spectrometer. This work investigated the effect of various instrument parameters including source temperatures, in-source CID, microscan type and quantity, resolution, and automatic gain control on spectral quality. The signal-to-noise ratio was found to be a suitable spectral attribute which facilitated identification of low abundance PTMs. Source temperature and CID voltage were found to require specific optimization for each protein. This study identifies key instrumental parameters requiring optimization for improved detection of a variety of PTMs by LC-MS and establishes a methodological framework to ensure robust proteoform identifications, the first step in their ultimate quantification.

Quantification of Total Vitamin-D-Binding Protein and the Glycosylated Isoforms by Liquid Chromatography-Isotope Dilution Mass Spectrometry.

Vitamin-D-binding protein (VDBP), a transporter of 25-hydroxyvitamin D metabolites, has three common isoforms. The relationship of the isoforms and their glycosylation state with various diseases has been under recent examination. In this work, liquid chromatography coupled to isotope dilution mass spectrometry was evaluated for quantification of VDBP, the three common isoforms, and total glycosylation. Protocols using guanidine, urea, RapiGest, trifluoroethanol, or tris buffer were also evaluated for optimal tryptic digestion. Differences in peptide release were detected between purified and plasma VDBP; however, for both protein sources, ELPEHTVK, TSALSAK, and VLEPTLK concentrations were reproducible between most protocols tested. The isoform-specific peptides, LPDATPK, LPDATPTELAK, and LPEATPTELAK, were optimally released when TFE was added to plasma. The total VDBP concentration calculated from the three shared peptides resulted in 97.6% accuracy compared with the concentration from amino acid analysis. Glycosylation of VDBP was also calculated for purified protein and donor samples using the ratio of the isoform-specific peptide(s) to the total protein concentration. Glycosylation of purified VDBP was found to be 99.5-111.1% the value determined by semiquantitative analysis of the intact protein by LC-MS. This approach may be used to quantify other samples containing a mixture of isoforms and post-translational modifications.

Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs.

N-glycosylation of proteins is well known to occur at asparagine residues that fall within the canonical consensus sequence N-X-S/T but has also been identified at a small number of asparagine residues within N-X-C motifs, including the N491 residue of human serotransferrin. Here we report novel glycosylation sites within noncanonical consensus motifs, in the conformation N-X-C, based on mass spectrometry analysis of partially deglycosylated glycopeptide targets. Alpha-1-acid glycoprotein (A1AG) and serotransferrin (Tf) were observed for the first time to be N-glycosylated on asparagine residues within a total of six unique noncanonical motifs. N-glycosylation was initially predicted in silico based on the evolutionary conservation of the N-X-C motif among related mammalian species and demonstrated experimentally in A1AG from porcine, canine, and feline sources and in human serotransferrin. High-resolution liquid chromatography-tandem mass spectrometry was employed to collect fragmentation data of predicted GlcNAcylated peptides and to assign modification sites within N-X-C motifs. A combination of targeted analytical techniques that includes complementary mass spectrometry platforms, enzymatic digestions, and partial-deglycosylation procedures was developed to confirm the novel observations. Additionally, we found that A1AG in porcine and canine sources is highly N-glycosylated at a noncanonical motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis-the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of N-X-C motif N-glycosylation are relatively uncommon in the literature, this work adds to a growing list of glycoproteins reported with glycosylation at various forms of noncanonical motifs.

Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA).

This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.

Recent progress in metabolomics and the development of increasingly sensitive analytical techniques have renewed interest in global profiling, i.e., semiquantitative monitoring of all chemical constituents of biological fluids. In this work, we have performed global profiling of NIST SRM 1950, "Metabolites in Human Plasma", using GC-MS, LC-MS, and NMR. Metabolome coverage, difficulties, and reproducibility of the experiments on each platform are discussed. A total of 353 metabolites have been identified in this material. GC-MS provides 65 unique identifications, and most of the identifications from NMR overlap with the LC-MS identifications, except for some small sugars that are not directly found by LC-MS. Also, repeatability and intermediate precision analyses show that the SRM 1950 profiling is reproducible enough to consider this material as a good choice to distinguish between analytical and biological variability. Clinical laboratory data shows that most results are within the reference ranges for each assay. In-house computational tools have been developed or modified for MS data processing and interactive web display. All data and programs are freely available online at http://peptide.nist.gov/ and http://srmd.nist.gov/ .

Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses.

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Detection, identification, and quantification of selenoproteins in a candidate human plasma standard reference material.

To understand the effect of Se supplementation on health, it is critical to accurately assess the Se status in the human body by measuring reliable biomarkers. The preferred biomarkers of the Se status are selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) along with selenoalbumin (SeAlb), but there is still a real need for reference methods and reference materials to validate their measurements. Therefore, this work presents a systematic approach to provide quality control data in selenoprotein measurements. This approach combines online isotope dilution affinity liquid chromatography (LC) coupled to inductively coupled plasma mass spectrometry (ICPMS), laser ablation ICPMS, and tandem mass spectrometry (MS/MS) to identify and quantify SelP, GPx3, and SeAlb in a human plasma reference material SRM 1950. Quantitative determinations of SelP, GPx3, and SeAlb were 50.2 ± 4.3, 23.6 ± 1.3, and 28.2 ± 2.6 ng g(-1) as Se, respectively. The subsequent identification of the selenoproteins included nine SelP peptides, including two selenopeptides and nine GPx3 peptides, while albumin was identified with a protein coverage factor >95%. The structural elucidation of selenoproteins in the target Se affinity fractions in SRM 1950 provides information needed for method validation and quality control measurements of selenoproteins and therefore the selenium status in human plasma.

Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Assessing a method and reference material for quantification of vitamin D binding protein during pregnancy.

Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.

Characterization of a human liver reference material fit for proteomics applications.

The National Institute of Standards and Technology (NIST) is creating new, economical, qualitative reference materials and data for proteomics comparisons, benchmarking and harmonization. Here we describe a large dataset from shotgun proteomic analysis of RM 8461 Human Liver for Proteomics, a reference material being developed. Consensus identifications using multiple search engines and sample preparations demonstrate a homogeneous and fit-for-purpose material that can be incorporated into automated or manual sample preparation workflows, with the resulting data used to directly assess complete sample-to-data workflows and provide harmonization and benchmarking between laboratories and techniques. Data are available via PRIDE with identifier PXD013608.

Selenium protein identification and profiling by mass spectrometry: A tool to assess progression of cardiomyopathy in a whale model.

Non-ischemic cardiomyopathy is a leading cause of congestive heart failure and sudden cardiac death in humans and in some cases the etiology of cardiomyopathy can include the downstream effects of an essential element deficiency. Of all mammal species, pygmy sperm whales (Kogia breviceps) present the greatest known prevalence of cardiomyopathy with more than half of examined individuals indicating the presence of cardiomyopathy from gross and histo-pathology. Several factors such as genetics, infectious agents, contaminants, biotoxins, and inappropriate dietary intake (vitamins, selenium, mercury, and pro-oxidants), may contribute to the development of idiopathic cardiomyopathy in K. breviceps. Due to the important role Se can play in antioxidant biochemistry and protein formation, Se protein presence and relative abundance were explored in cardiomyopathy related cases. Selenium proteins were separated and detected by multi-dimension liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), Se protein identification was performed by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS), and Se protein profiles were examined in liver (n=30) and heart tissue (n=5) by SEC/UV/ICP-MS detection. Data collected on selenium proteins was evaluated in the context of individual animal trace element concentration, life history, and histological information. Selenium containing protein peak profiles varied in presence and intensity between animals with no pathological findings of cardiomyopathy and animals exhibiting evidence of cardiomyopathy. In particular, one class of proteins, metallothioneins, was found to be associated with Se and was in greater abundance in animals with cardiomyopathy than those with no pathological findings. Profiling Se species with SEC/ICP-MS proved to be a useful tool to identify Se protein pattern differences between heart disease stages in K. breviceps and an approach similar to this may be applied to other species to study Se protein associations with cardiomyopathy.

Multiple forms of selenoprotein P in a candidate human plasma standard reference material.

Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.

Formation of y + 10 and y + 11 ions in the collision-induced dissociation of peptide ions.

Tandem mass spectra of peptide ions, acquired in shotgun proteomic studies of selected proteins, tissues, and organisms, commonly include prominent peaks that cannot be assigned to the known fragmentation product ions (y, b, a, neutral losses). In many cases these persist even when creating consensus spectra for inclusion in spectral libraries, where it is important to determine whether these peaks represent new fragmentation paths or arise from impurities. Using spectra from libraries and synthesized peptides, we investigate a class of fragment ions corresponding to y(n-1) + 10 and y(n-1) + 11, where n is the number of amino acid residues in the peptide. These 10 and 11 Da differences in mass of the y ion were ascribed before to the masses of [+ CO - H(2)O] and [+ CO - NH(3)], respectively. The mechanism is suggested to involve dissociation of the N-terminal residue at the CH-CO bond following loss of H(2)O or NH(3). MS(3) spectra of these ions show that the location of the additional 10 or 11 Da is at the N-terminal residue. The y(n-1) + 10 ion is most often found in peptides with N-terminal proline, asparagine, and histidine, and also with serine and threonine in the adjacent position. The y(n-1) + 11 ion is observed predominantly with histidine and asparagine at the N-terminus, but also occurs with asparagine in positions two through four. The intensities of the y(n-1) + 10 ions decrease with increasing peptide length. These data for y(n-1) + 10 and y(n-1) + 11 ion formation may be used to improve peptide identification from tandem mass spectra.

Changes in protein expression in Burkholderia vietnamiensis PR1 301 at pH 5 and 7 with and without nickel.

Burkholderia vietnamiensis PR1(301) (PR1) exhibits pH-dependent nickel (Ni) tolerance, with lower Ni toxicity observed at pH 5 than at pH 7. The Ni tolerance mechanism in PR1 is currently unknown, and traditional mechanisms of Ni resistance do not appear to be present. Therefore, 2D gel electrophoresis was used to examine changes in protein expression in PR1 with and without Ni (3.4 mM) at pH 5 and 7. Proteins with both a statistically significant and at least a twofold difference in expression level between conditions (pH, Ni) were selected and identified using MALDI-TOF-MS or LC-MS. Results showed increased expression of proteins involved in cell shape and membrane composition at pH 5 compared with pH 7. Scanning electron microscopy indicated elongated cells at pH 5 and 6 compared with pH 7 in the absence of Ni. Fatty acid methyl ester analysis showed a statistically significant difference in the percentages of long- and short-chain fatty acids at pH 5 and 7. These findings suggest that changes in membrane structure and function may be involved in the ability of PR1 to grow at higher concentrations of Ni at pH 5 than at pH 7.