Lisa E. Kilpatrick

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.

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.

A proteomics performance standard to support measurement quality in proteomics.

The emergence of MS‐based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high‐quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics‐based experimental workflows. RM 8323 yeast protein extract is based upon the well‐characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics‐based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC‐MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC‐MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics‐based sample preparation methods.

Detection and characterization of selenoproteins by tandem mass spectrometry assisted by laser ablation inductively coupled plasma mass spectrometry: application to human plasma selenoproteins

Accurate assessment of selenium status and selenium nutritional requirement in humans through the detection, identification and quantification of human plasma selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) has been an on-going effort and long term goal. Although several HPLC-ICP-MS analyses of human plasma/serum have reported SelP and GPx3 measurements, none of them have yet to demonstrate unambiguous mass spectrometry-based identification of these proteins. This study explored the potential of mass spectrometry techniques for the detection and identification of selenoproteins in a human plasma candidate Standard Reference Material (SRM) 1950 Metabolites in Human Plasma, with a total selenium concentration of 105.5 ± 2.3 ng g−1. Since the classical proteomic shotgun approach of depleted human plasma is not specific and sensitive enough to identify low abundant selenoproteins, a laser ablation (LA) inductively coupled plasma mass spectroscopy (ICP-MS) method was developed. The challenge was to develop a highly sensitive method to target selenoproteins at physiological concentrations by using a conventional laser device and a sensitive LA-ICP-MS method easily transposable to other equipped laboratories. Through a combination of better sample preparation, by concentrating selenoproteins onto membranes, and increased sensitivity of Se detection, by humidifying ICP with an organic solution, a LA-ICP-MS method 80 times more sensitive compared to classical LA ICP-MS methods was developed. This method was successfully applied to the detection of SelP and GPx3 selenoproteins at physiological concentrations in samples of human plasma. Once detected, these low abundant selenoproteins were unambiguously identified by tandem mass spectrometry. This study highlights the importance of an approach combining ICP-MS and tandem mass spectrometry for unequivocal selenoprotein detection and identification at physiological concentrations. This procedure can be easily performed in other laboratories to study selenoproteins or other heteroatom-tagged proteins in humans or other biological samples using widely available instrumentation.

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 (HSA1) 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.

Changes in protein expression in Burkholderia vietnamiensis PR1301 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.

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.

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.

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.