Comparison of spatio-temporal gait parameters between the GAITRite® platinum plus classic and the GAITRite® CIRFACE among older adults: a retrospective observational study.

BACKGROUND: The GAITRite® system is one of the gold standards for gait electronic analysis, especially for older adults. Previous GAITRite® systems were composed of an electronic roll-up walkway. Recently, a new GAITRite® electronic walkway, named CIRFACE, was commercialized. It is composed of a changeable association of stiff plates, unlike previous models. Are the gait parameters measured similar between these two walkways among older adults and according to the cognitive status, the history of falls, and the use of walking aids? METHODS: In this retrospective observational study, 95 older ambulatory participants (mean, 82.6 ± 5.8 years) were included. Ten spatio-temporal gait parameters were measured simultaneously with the two GAITRite® systems in older adults while walking at comfortable self-selected pace. The GAITRite® Platinum Plus Classic (26') was superimposed on the GAITRite® CIRFACE (VI). Comparisons between the parameters of the two walkways were performed using Bravais-Pearson correlation, between-method differences (corresponding to bias), percentage errors and Intraclass Correlation Coefficients (ICC2,1). Subgroup analyses were performed according to the cognitive status, the history of falls in the last 12 months and the use of walking aids. RESULTS: The whole walk parameters recorded by the two walkways were extremely correlated with a Bravais-Pearson correlation coefficient ranging from 0.968 to 0.999, P < .001, indicating a very high correlation. According to the ICC2,1 calculated for absolute agreement, all gait parameters had excellent reliability (ranging from 0.938 to 0.999). Mean bias for 9 parameters out of 10 were ranged from - 0.27 to 0.54, with clinically acceptable percentage errors (1.2-10.1%). Step length showed a substantially higher bias (1.4 ± 1.2 cm), nevertheless the percentage errors remained clinically acceptable (5%). CONCLUSION: When walking at comfortable self-selected pace, the standard spatio-temporal walk parameters provided by both the GAITRite® PPC and the GAITRite® CIRFACE seem similar and very highly correlated in older adults with various cognitive or motor status. The data of studies using these systems can be compared and mixed with a very low risk of bias in a meta-analytic process. Also, the geriatric care units can choose the most ergonomic system according to their infrastructure without affecting their gait data. TRIAL REGISTRATION: NCT04557592 (21/09/2020).

Physical Activity and Fall Prevention in Geriatric Inpatients in an Acute Care Unit (AGIR Study): Protocol for a Usability Study.

BACKGROUND: Falls are one of the world's top 10 risks associated with disability in people older than 60 years. They also represent more than two-thirds of adverse events in hospitals, mainly affecting patients older than 65 years. Physical activity is a central intervention in fall prevention for older people. Whatever the details of the prevention strategy that is adopted (ie, how a mono- or multifactorial intervention is evaluated, the category of person the intervention targets, and where it is used), it is important to ensure that the proposed intervention is feasible and usable for the patient and the health care team. OBJECTIVE: The primary objective is to study the usability of carrying out a physical activity intervention, including 3 types of exercises, in older patients hospitalized in a geriatric acute care unit and categorized according to 3 fall risk levels: low, moderate, and high. The secondary objectives are to determine the difficulty of the physical exercise for patients with different fall risk levels, to study the health care team's perceptions of the intervention's feasibility, and to study the benefits for patients. METHODS: This is an open-label, unicenter, nonrandomized, usability prospective clinical trial. The intervention tested is a daily physical activity program. It consists of 3 types of physical exercise: staying out of bed for at least 3 hours, performing balance exercises while standing for 2 minutes, and the Five Times Sit to Stand transfer exercise. These exercises are carried out under the supervision of the health care team. Fall risk in the patients is classified with the Brief Geriatric Assessment tool. The exercise program starts on the second day of hospitalization after inclusion in the study. Patient assessment continues until the last day of hospitalization or the 20th day of hospitalization, whichever is earlier. For each fall-risk group and each type of exercise, the intervention will be defined as usable if at least 80% of the participants complete 75% or more of the exercises (ie, the ratio between the number of days when the patient completes a type of exercise and the total number of hospitalization days). The perceived feasibility by the health care team is measured with 2 scales, measuring perceived difficulty and time spent with the patient. The intervention benefit is evaluated using the performance of the Five Times Sit to Stand test before and after the intervention. RESULTS: The first patient was recruited on March 16, 2015. The study enrolled 266 patients, including 75 with low fall risk, 105 with moderate risk, and 85 with high risk. CONCLUSIONS: We have not yet analyzed the results, but our observations suggest that the usability of each type of exercise for a given patient will depend on their fall risk level. TRIAL REGISTRATION: ClinicalTrials.gov NCT02393014; https://clinicaltrials.gov/ct2/show/NCT02393014. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32288.

Vitamin D supplementation prior to or during COVID-19 associated with better 3-month survival in geriatric patients: Extension phase of the GERIA-COVID study.

BACKGROUND: The objective of this extension phase of the quasi-experimental GERIA-COVID study was to determine whether vitamin D3 supplementation taken prior to or during COVID-19 was associated with better 3-month survival in geriatric patients hospitalized for COVID-19. METHODS: Intervention group was defined as all participants supplemented with vitamin D3 prior to or during COVID-19 (n = 67). Supplements were either bolus vitamin D3 (ie, 50,000 IU per month, or 80,000 IU or 100,000 IU or 200,000 IU every 2-3 months), or daily supplementation with 800 IU. Comparator group involved those without vitamin D supplements (n = 28). Outcome was 3-month mortality. Covariables were age, sex, functional abilities, history of malignancies, cardiomyopathy, undernutrition, number of acute health issues, antibiotics use, systemic corticosteroids use, and 25(OH)D concentration. RESULTS: 76.1 % (n = 51) of participants survived at 3 months in Intervention group, compared to only 53.6 % (n = 15) in Comparator group (P = 0.03). The fully-adjusted hazard ratio for 3-month mortality was HR = 0.23 [95 %CI: 0.09;0.58](P = 0.002) in Intervention group compared to Comparator group. Intervention group had also longer survival time (log-rank P = 0.008). CONCLUSIONS: Vitamin D3 supplementation was associated with better 3-month survival in older COVID-19 patients.

COvid-19 and high-dose VITamin D supplementation TRIAL in high-risk older patients (COVIT-TRIAL): study protocol for a randomized controlled trial.

BACKGROUND: With the lack of effective therapy, chemoprevention, and vaccination against SARS-CoV-2, focusing on the immediate repurposing of existing drugs gives hope of curbing the COVID-19 pandemic. A recent unbiased genomics-guided tracing of the SARS-CoV-2 targets in human cells identified vitamin D among the three top-scoring molecules manifesting potential infection mitigation patterns. Growing pre-clinical and epidemiological observational data support this assumption. We hypothesized that vitamin D supplementation may improve the prognosis of COVID-19. The aim of this trial is to compare the effect of a single oral high dose of cholecalciferol versus a single oral standard dose on all-cause 14-day mortality rate in COVID-19 older adults at higher risk of worsening. METHODS: The COVIT-TRIAL study is an open-label, multicenter, randomized controlled superiority trial. Patients aged ≥ 65 years with COVID-19 (diagnosed within the preceding 3 days with RT-PCR and/or chest CT scan) and at least one worsening risk factor at the time of inclusion (i.e., age ≥ 75 years, or SpO2 ≤ 94% in room air, or PaO2/FiO2 ≤ 300 mmHg), having no contraindications to vitamin D supplementation, and having received no vitamin D supplementation > 800 IU/day during the preceding month are recruited. Participants are randomized either to high-dose cholecalciferol (two 200,000 IU drinking vials at once on the day of inclusion) or to standard-dose cholecalciferol (one 50,000 IU drinking vial on the day of inclusion). Two hundred sixty participants are recruited and followed up for 28 days. The primary outcome measure is all-cause mortality within 14 days of inclusion. Secondary outcomes are the score changes on the World Health Organization Ordinal Scale for Clinical Improvement (OSCI) scale for COVID-19, and the between-group comparison of safety. These outcomes are assessed at baseline, day 14, and day 28, together with the serum concentrations of 25(OH)D, creatinine, calcium, and albumin at baseline and day 7. DISCUSSION: COVIT-TRIAL is to our knowledge the first randomized controlled trial testing the effect of vitamin D supplementation on the prognosis of COVID-19 in high-risk older patients. High-dose vitamin D supplementation may be an effective, well-tolerated, and easily and immediately accessible treatment for COVID-19, the incidence of which increases dramatically and for which there are currently no scientifically validated treatments. TRIAL REGISTRATION: ClinicalTrials.gov NCT04344041 . Registered on 14 April 2020 TRIAL STATUS: Recruiting. Recruitment is expected to be completed in April 2021.

Communication Technology Preferences of Hospitalized and Institutionalized Frail Older Adults During COVID-19 Confinement: Cross-Sectional Survey Study.

BACKGROUND: Technological communication methods such as telephone calls and video calls can help prevent social isolation and loneliness in frail older adults during confinement. OBJECTIVE: Our objectives were to determine which virtual communication method (ie, telephone call or video call) was preferred by confined older hospital patients and nursing home residents and the variables influencing this preference. METHODS: The TOVID (Telephony Or Videophony for Isolated elDerly) study was a cross-sectional study that was designed to examine the preference between telephone calls and video calls among frail older adults who were either hospitalized in a geriatric acute care unit or institutionalized in a long-term care and nursing home during the COVID-19 confinement period. RESULTS: A total of 132 older people were surveyed between March 25 and May 11, 2020 (mean age 88.2 years, SD 6.2); 79 (59.8%) were women. Patients hospitalized in the geriatric acute care unit were more able to establish communication independently than residents institutionalized in the long-term care and nursing home (P=.03) and were more satisfied with their communication experiences (P=.02). Overall, older people tended to favor telephone calls (73/132, 55.3%) over video calls (59/132, 44.7%); however, their satisfaction degree was similar regardless of the chosen method (P=.1), with no effect of age (P=.97) or gender (P=.2). In the geriatric acute care unit, the satisfaction degrees were similar for telephone calls (40/41, 98%) and video calls (33/38, 87%) in older patients (P=.10). Conversely, in the long-term care and nursing home, residents were more satisfied with the use of video calls to communicate with their relatives (14/15, 93%) versus the use of telephone calls (6/12, 50%; P=.02). CONCLUSIONS: Older people confined to health care settings were able to complete telephone calls more independently than video calls, and they tended to use telephone calls more often than video calls. The satisfaction degrees were similar with both modalities and even greater with video calls among long-term care and nursing home residents when they were given assistance to establish communication. TRIAL REGISTRATION: ClinicalTrials.gov NCT04333849: https://www.clinicaltrials.gov/ct2/show/NCT04333849.

Serum Vitamin D and Cingulate Cortex Thickness in Older Adults: Quantitative MRI of the Brain.

BACKGROUND: Vitamin D insufficiency is associated with brain changes, and cognitive and mobility declines in older adults. OBJECTIVE: Our objective was to investigate in older adults whether vitamin D insufficiency<50nmol/L was associated with thinner cingulate cortex, a brain area related to cognitive functions influenced by vitamin D. METHODS: Two hundred and fifteen Caucasian older community-dwellers (mean±SD, 72.1±5.5years; 40% female) received a blood test and brain MRI. The thickness of perigenual anterior cingulate cortex, midcingulate cortex and posterior cingulate cortex was measured using FreeSurfer from T1-weighted MR images. Age, gender, education, BMI, mean arterial pressure, comorbidities, use of vitamin D supplements or anti-vascular drugs, MMSE, GDS, IADL, serum calcium and vitamin B9 concentrations, creatinine clearance were used as covariables. RESULTS: Participants with vitamin D insufficiency (n=80) had thinner total cingulate thickness than the others (24.6±1.9mm versus 25.3±1.4mm, P=0.001); a significant difference found for all 3 regions. Vitamin D insufficiency was cross-sectionally associated with a decreased total cingulate thickness (ß=- 0.49, P=0.028). Serum 25OHD concentration correlated positively with the thickness of perigenual anterior (P=0.011), midcingulate (P=0.013) and posterior cingulate cortex (P=0.021). CONCLUSION: Vitamin D insufficiency was associated with thinner cingulate cortex in the studied sample of older adults. These findings provide insight into the pathophysiology of cognitive and mobility declines in older adults with vitamin D insufficiency.

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Absolute quantification of podocalyxin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-mass spectrometry.

Podocalyxin is a protein present in specialized glomerulus cells called podocytes and may be released in the urine in case of kidney injury. In this context, its quantification could be of great interest in order to monitor glomerular injury. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) mode, has been demonstrated as a powerful technique that can be applied to protein quantification. This paper describes the development of a quantification method of human podocalyxin in urine by LC-MS/MS in SRM mode by monitoring one proteotypic peptide with an isotope-dilution standardization strategy employing (13)C/(15)N labelled peptides. Inter/intra assay precisions and accuracies of the assay were below 10% and between 90% and 106.1%, respectively. In addition, the method was linear between 0.78 and 100 ng/mL and could therefore be used to quantify endogenous level of podocalyxin that was estimated between 15.2 and 44.2 ng/mL in urine samples from healthy donor.

Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.

Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF. Development involved peptide transition optimization, denaturation/digestion protocol evaluation, transition interference screening, and protein quantitation via peptide standard curves. The final method exhibited excellent reproducibility (average coefficient of variation of <1% for retention time and <6% for signal) and breadth of quantitation (130 proteins from 311 interference-free peptides) in a single 43-min run. These proteins are of high-to-low abundance with determined concentrations from 118 µg/mL (serum albumin) to 550 pg/mL (apolipoprotein C-I). Overall, the method consists of the most highly multiplexed and broadest panel of candidate protein biomarkers in human CSF reported thus far and is well suited for subsequent verification studies on patient samples.

Hydrophilic interaction liquid chromatography as second dimension in multidimensional chromatography with an anionic trapping strategy: application to prostate-specific antigen quantification.

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS-MS) in selected reaction monitoring mode (SRM) has become a widely used technique for the quantification of protein biomarkers in plasma and has already proven to give similar results compared to the conventional immunoassays. To improve the lack of insufficient sensitivity for quantification of low abundance protein, we propose a new two dimensional liquid chromatography (2D-LC-SRM) method for the quantitation of prostate specific antigen (PSA) in human plasma. The method centers on anion exchange cartridge between reversed-phase chromatography and hydrophilic interaction liquid chromatography (HILIC) in an on-line arrangement. The use of the anionic cartridge allows an easier online transfer of the analytes between both dimensions. Moreover, it provides an additional selectivity since the more basic peptides are not retained on this support. This setup has been applied to the quantification of prostate specific antigen (PSA) protein in plasma on a previous generation of mass spectrometer, which enabled a limit of quantification (LOQ) of 1ng/mL without any upfront immuno-depletion or intense off-line fractionation before the SRM analysis. The obtained LOQ is compatible with the required sensitivity for the clinically relevant plasma-based PSA tests.

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.

Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples. BIOLOGICAL SIGNIFICANCE: The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies.

Absolute quantification of podocin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-multiple reaction monitoring cubed mass spectrometry.

Glomeruli play a major role in the kidney function since they are involved in primary urine formation. It is then crucial to dispose of methods to monitor glomerular injury, especially in drug development. In this context, quantification of podocin could be of great interest since it is a protein exclusively present in highly specialized glomerulus cells called podocytes. Immunoassays are the most commonly used approach for protein assays. However, they rely on the availability of specific antibodies. When such antibodies are not available, liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) or in multiple reaction monitoring cubed (MRM(3)) mode, has been demonstrated as a powerful alternative technique, and can be applied to multiple protein quantification. This paper describes the development of a quantification method of human podocin in urine by LC-MS/MS in MRM(3) mode. Inter assay precision and accuracy ranged from 7 to 20% and from 105 to 112% respectively and the lower limit of quantification (LLOQ) was 0.39ng/mL from only 1mL of urine which is compatible for endogenous level of podocin determination.

MRM for the verification of cancer biomarker proteins: recent applications to human plasma and serum.

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 µg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

Combined collision-induced dissociation and photo-selected reaction monitoring mass spectrometry modes for simultaneous analysis of coagulation factors and estrogens.

Oral estrogens are directly associated with changes in plasma levels of coagulation proteins. Thus, the detection of any variation in protein concentrations due to estrogen contraceptives, by a simultaneous analysis of both coagulation proteins and estrogens, would be a very informative tool. In the present study, the merit of photo-selected reaction monitoring (SRM), a new analytical tool, was evaluated towards estrogens detection in plasma. Then, SRM and photo-SRM detection modes were combined for the simultaneous analysis of estrogen molecules together with heparin co-factor and factor XIIa, two proteins involved in the coagulation cascade. This study shows that photo-SRM could open new multiplexed analytical routes.

Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine.

Aquaporin-2 (AQP2) is a water channel protein located in the kidney collecting ducts that has been studied as a potential biomarker of a wide variety of water handling disorders and that could also be used to monitor lesions in the collecting ducts. Enzyme-linked immunosorbent assay (ELISA), the most commonly used approach for protein assay in biofluids, has a limited potential for biomarker verification due to the restricted possibility to perform multiplex assays, the cost and complexity of assay development for new candidates. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in multiple reaction monitoring (MRM) mode, has been demonstrated as a powerful alternative technique, and applied to multiple protein quantification. An even more specific method, termed MRM cubed (MRM(3)), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC-MS/MS, based on the MRM(3) strategy, and the influence of key MRM(3) parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5-50ng/mL, intra and inter assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%. This assay could therefore be used in the near future to evaluate human urinary AQP2 as a potential biomarker of kidney collecting duct injury.

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM.

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Evaluation of hydrophilic interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted quantification of peptides by mass spectrometry.

Hydrophilic-interaction liquid chromatography (HILIC) is a widely used technique for small polar molecule analysis and offers the advantage of improved sensitivity in mass spectrometry. Although HILIC is today frequently employed as an orthogonal fractionation method for peptides during the proteomic discovery phase, it is still seldom considered for quantification. In this study, the performances in terms of peak capacity and sensitivity of 3 HILIC columns were compared to traditional reversed phase liquid C(18) column in the context of targeted quantification of proteotypic peptides using selected reaction monitoring mode (SRM). The results showed that the maximum sensitivity in HILIC chromatography was achieved by using an amide column without salt buffer and that the signal increased compared to classic reversed phase chromatography. However, the intensity improvement is quite low compared to the one obtained for small molecules. This is due on one hand to a higher matrix effect in HILIC and on the other hand to a change of charge states of peptides in organic solvent (doubly charged to monocharged). The doubly charged ions can be more readily dissociated than singly charged ions, making them ideal for SRM peptide quantification. As a result "supercharging" reagents are added to the mobile phase to shift from predominant singly charged ions to the more favorable doubly charged species. Using such optimized conditions, peptide signal is improved by a factor of between two and ten for 88% of the peptides of the 81 peptides investigated.

Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides.

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n=68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n=669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.