the Biology of Apolipoprotein M

Global LC-MS/ MS Analysis of Human and Rat Serum or Plasma Global profiling studies were used to evaluate the entire protein content of each sample using a triple-play method to collect

INTRODUCTION

Introduction to the Biology of Apolipoprotein M (ApoM)

Cholesterol is an integral part of cell membranes and in the synthesis of steroids, bile, and vitamin D Cholesterol found in the human body comes from two sources: biosynthesis and diet [1] Cholesterol is a hydrophobic molecule and cannot travel

through the bloodstream without assistance from lipoproteins Lipoprotein particles consist of protein components called apolipoproteins that help solubilize the hydrophobic lipids [2] Apolipoproteins transport cholesterol through the bloodstream to target organs and tissues A schematic of cholesterol transport and cycling of lipoproteins is shown in Figure 1 [3] Lipoprotein particles are generally classified as high, intermediate, low, and very low density lipoproteins (HDL, IDL, LDL, and VLDL, respectively), and

chylomicrons [1] Lipid-free apolipoprotein AI and lipid-poor preβ-HDL particles are precursors to the generation of mature, lipid-containing HDL particles Apolipoprotein

M is a component of preβ-HDL and is present in a sub-population of mature HDL

particles Preβ-HDL particles are thought to function primarily to induce cholesterol efflux from cells and act as the initial acceptors of cholesterol from peripheral tissues in reverse cholesterol transport (RCT) [2] In RCT, HDL accepts cholesterol from

peripheral cells, transports, and delivers it to the liver for degradation and excretion [2] Preβ-HDL accepts unesterfied cholesterol and phospholipids, then the enzyme LCAT (lecithin cholesterol acyltransferase) esterfies the free cholesterol to modify the preβ-HDL into the spherical α-HDL (cholesterol ester core) [3] The pre-beta form of HDL

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exists in a lipid-poor state and may be indicative of the cholesterol-carrying capacity of the HDL in circulation

Human apoM was discovered by Xu, N et al in 1999 [4] and is mainly associated

with a small sub-population of HDL particles [5] HDL particles lacking apoM showed a 50% decrease in cholesterol efflux from macrophages compared to apoM-containing

HDL particles in vitro [2] Human apoM is primarily associated with lipid-poor HDL, mature HDL, and to a lesser extent LDL and VLDL [4] Wolfrum et al

preβ-demonstrated that apoM is necessary for the formation of these preβ-HDL particles [2]

It has been shown that elevated levels of LDL, VLDL, and chylomicrons in circulation can lead to atherosclerotic lesion formation, whereas HDL has protective anti-atherogenic effects [6] As a component of HDL, apoM is thought to have a significant influence on the development of coronary heart disease (CHD), caused by atherosclerosis [7] Atherosclerotic lesions are a product of the accumulation of macrophage foam cells

in blood vessels Foam cells can be formed when excess LDL in circulation is oxidized and engulfed by macrophages [8] Accumulation of chylomicron remnants in

macrophages can also promote foam cell formation without oxidation [9] The apoM present in LDL particles is thought to play a protective role against CHD by reducing oxidation of LDL and it has been shown that apoM-containing LDL particles are more resistant to oxidation [10] LDL receptor knockout (ldlr-/-) mice over-expressing apoM had 70% less atherosclerotic lesion formation compared to ldlr-/- mice with endogenous normal apoM levels [2], supporting the anti-atherogenic benefits of apoM and making it a significant apolipoprotein in the study of CHD

ApoM is also studied in association with other lipid metabolic diseases including diabetes, specifically mature-onset diabetes of the young (MODY3) [11, 12] and obesity [13] Since apoM is a recently characterized protein [4], a quantitative assay for apoM has not yet been well established Western blots have been used to demonstrate changes

in apoM levels [2], but this method is not highly quantitative and suffers from low

throughput To date there is not a commercially-available quantitative assay for apoM in any species The ability to quantitatively analyze apoM will enable a better

understanding of its behavior in relationship with certain diseases Even though

development and use of an ELISA has been reported [14], the assay is not widely

available In-house efforts by a collaborator to replicate an ELISA measuring apoM in human serum has proven difficult and was ultimately unsuccessful (unpublished data)

To address the need for a quantitative assay for apoM in serum, an antibody-free, high throughput, mass spectrometry-based assay was developed

Introduction to Mass Spectrometry

The use of high performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) has been gaining popularity for development of quantitative assays over the past five years [15] However, this approach often requires an antibody for selective enrichment of the target protein, especially when the protein of interest is in low abundance [16] We developed a MS-based targeted assay to quantify apoM in human and rodent (mouse or rat) serum that does not require the use of an antibody This label-free method to quantify apoM in serum provides a powerful tool to further understand the biology of apoM with many research applications

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In the initial stages of the development of a quantitative assay for a specific

protein or proteins using a targeted LC-MS method, an unbiased global MS approach can

be used to identify the target protein in serum Global profiling studies are typically done

to identify the entire protein content of a sample Global profiling of a biological sample (i.e serum) begins with enzymatic digestion of the proteins, typically with trypsin to create tryptic peptides These peptides can be separated based on hydrophobicity using a simple, two and a half hour HPLC gradient prior to MS analysis The peptides in the effluent of the HPLC column are ionized and sprayed into an on-line ion trap MS, such as

an LTQ (Thermo) Proteins are identified from this analysis based on the identification

of unique tryptic peptides from a specific protein

The ionized tryptic peptides are measured by MS as a mass-to-charge ratio (m/z)

using the molecular mass and charge status of each peptide In a global profiling study, a triple-play method can be used to collect ion spectra using three MS scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan

The full MS scan captures the mass-to-charge (m/z) ratios of all ionized peptides eluted at

a specific time point The most abundant peptide in the scan is selected as a precursor ion and a zoom scan of this peptide is used to estimate the charge status and monoisotopic and average masses of the peptide The zoom scan also evaluates the quality of the selected peptide to avoid false positive protein identifications and eliminates low quality data from further analysis The selected peptide is then fragmented, and the spectra of these product ions are collected by a MS/ MS scan [17] The product ions from

fragmentation of the precursor ion at the amide bond of the peptide backbone are

classified as b-ions and y-ions A b-ion is observed when the proton is retained by the

N-terminal fragment of the peptide, and a y-ion is observed with the retention of the proton

by the C-terminal fragment of the peptide [18] The fragmentation spectra is searched against species-specific computerized protein databases and used for peptide sequencing and protein identification

The data output from the database search yields protein identifications based on the detection of a tryptic peptide that is unique to the protein from which it was derived

A targeted MS method can be created for a specific protein or proteins of interest using the results from these global profiling studies Identification of the protein of interest and the MS/ MS spectra generated from the fragmentation of the precursor ion are collected

by the global studies and are used to set up a targeted Multiple Reaction Monitoring (MRM) assay using the same instrument [19]

The m/z values of the precursor ion and respective fragmentation y- and b-ions

obtained from the global profiling studies are used to set up a targeted assay to measure

only specific m/z values Only these m/z values will be collected by the MS while the m/z values of precursor ions that do not fall within the set m/z window are filtered out The

MS peptide signal from the precursor ion can be integrated to obtain the area-under-curve value (AUC) which can be used to quantify the target protein

These MS methods were used to develop a targeted assay for the quantification of apoM in human and rodent serum This assay spans multiple species to streamline the use of this assay between pre-clinical and clinical measurements The resulting assay is versatile and quantitative for apoM and will provide a powerful tool to expand research in CHD and other diseases and provide a deeper understanding of the biology of apoM in many different applications

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Figure 1

Figure 1: Lipoprotein involvement in lipid transport

The transport of lipid through the bloodstream to target cells is achieved by lipoproteins This schematic of lipid transport was from Brewer, HB Jr., N Engl J Med 2004;

350:1491-1494, Apr 8, 2004

Pel-by purification of the recombinant protein from E.coli grown in a medium containing

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N-urea Mouse anti-apoM primary antibody was from BD Transduction Labs (cat

#612333) and an ECC anti-mouse IgG-Horseradish peroxidase secondary antibody was

from Amersham (cat #NA931V) ECL kit was from Amersham (cat #RPN303D)

Human recombinant apoM from E.coli was grown in-house and given by Dr Thomas

Lee (Eli Lilly and Co.)

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Sample Preparation for Global LC-MS/ MS Analysis of Human and Rat Serum

Serum was enriched with lipoprotein-binding beads to selectively purify

apolipoproteins from serum prior to digestion with trypsin The digested proteins were separated by reverse-phase HPLC and analyzed by MS using a global profiling method to identify the entire protein content of each sample

Aliquots of human and rat serum were prepared with PHM-Liposorb in PBS to selectively remove lipoproteins from serum A method of lipoprotein removal using Liposorb has been described previously [20] and was adapted for selective removal of apolipoproteins from serum prior to MS analysis by Dr Bomie Han (Eli Lilly and Co.) Liposorb powder (one gram) was suspended in 50 mL PBS and filtered through a 250 µm filter The flow-through was used as the Liposorb working stock solution (1 g/ 50 mL) Ten microliters (10 µL) of serum was diluted in 90 µL of PBS and incubated with 100 µL

of Liposorb stock for 10 minutes at room temperature with shaking to keep Liposorb suspended The samples were spun down to pellet the Liposorb and supernatant was removed The pellet was washed three times with 500 µL of 100 mM ammonium

bicarbonate (ABC) and digested overnight at 37°C with 1 µg of modified trypsin prior to

MS analysis Samples were filtered and 50 µL of 1 mL final volume (0.5 µL of serum) was analyzed by triple-play LC-MS/ MS (LTQ from Thermo) in global profiling studies

Global LC-MS/ MS Analysis of Human and Rat Serum or Plasma

Global profiling studies were used to evaluate the entire protein content of each sample using a triple-play method to collect spectral data from the fragmentation of unique peptides to identify each respective protein The HPLC gradient was controlled using a Surveyor MS pump (Thermo) equipped with a sample loop and a Zorbax SB300-C18 (3.5 µm particle size) 1 mm x 50 mm reverse phase column (Agilent Technologies, cat #865630-902) The column was kept at 27°C and sample tray was 4°C during

analysis 50 µL of 1 mL total sample volume (0.5 µL of human or rat serum) was

injected into a 100 µL sample loop using the partial loop method The HPLC gradient ran for 142 minutes per injection and utilized a three buffer system [0.1% formic acid in

H2O (Buffer A), 0.1% formic acid in 50% acetonitrile (Buffer B), and 0.1% formic acid

in 80% acetonitrile (Buffer C)] The gradient ran at a 50 µL per minute flow rate of: 90%

A, 10% B from 0.0-5.0 min, then ran a sloped gradient from 90% A, 10% B to 5% A, 95% B from 5.0 to 125.0 min, then 100% C from 125.1-130.0 min, and 90% A, 10% B from 130.1 min-142.0 min This gradient separated the peptides based on

hydrophobicity The peptides were eluted, ionized, and sprayed directly into an online

MS for mass and charge state determination

Mass spectrometry data were collected in triple-play mode with three scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan Protein identification was performed using a Sequest and X! Tandem algorithm that combined the protein identifications output from each search [17] and these results were searched against a reverse database to confirm protein identifications The p-value

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of each tryptic peptide was used to determine the quality of the peptide identification to help avoid using false-positive identifications for method development

Sample Preparation for Targeted MS Analysis of ApoM

A heavy isotope-labeled apolipoprotein standard was spiked into each sample at the beginning of sample preparation to normalize variations in protein recovery from serum that may occur during sample preparation or instrument analysis Serum or plasma was enriched with lipoprotein-binding beads and the bound proteins were denatured with urea prior to digestion with trypsin in the presence of detergent

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N-labeled human apolipoprotein A-IV (15N-Apo A-IV) was used as an internal standard (iSTD) The labeled protein was diluted into PBS and 300 µL (250ng) was spiked into each experimental and external calibration sample Rabbit or horse serum was mixed with PBS (1:6) and used as a dilution matrix for human or mouse

experimental samples, respectively 10 µL of human or mouse serum was mixed with

140 µL of the dilution matrix to dilute the experimental sample in the background serum

at a 1:2 ratio (3x dilution of the experimental sample) The total serum-to-PBS ratio was 1:4 to mimic the ratio of total serum-to-PBS in the calibration samples 50 µL of the diluted sample was mixed into the internal standard solution 200 µL of Liposorb was used per 10 µL of serum Samples were incubated in Liposorb at 4°C for 20 minutes with shaking to keep Liposorb suspended

The Liposorb was spun down and supernatant aspirated The Liposorb pellet was washed one time with 100 mM ABC The washing step can also be done in a filter plate for higher throughput and will be described later The Liposorb pellet was resuspended

in either 100 µL of 8 M urea, or 10 mM ABC prior to reduction/ alkylation (R/A), with repeated pipetting of the sample in solution

In the urea-containing method, samples were incubated at room temperature for

15 minutes with shaking 200 µL of 100 mM ABC was added In an alternative

protocol, proteins were reduced with 100 µL of 10 mM dithiothreitol (DTT) at 37°C for

45 minutes and alkylated with 100 µL of 60 mM iodoacetamide at room temperature for

30 minutes In both protocols, modified trypsin was added at 2 µg in 200 µL of 0.1% NP40 in 100 mM ABC Samples were incubated at 37°C overnight with shaking Peptides were eluted from the Liposorb directly with digestion Samples were filtered after digestion to remove Liposorb from the final sample 50 µL of 500 µL final sample volume (1 µL serum) was injected to a 100 µL sample loop for the MS measurement of apoM

Preparation of External Calibration Standards

A series of external calibration standards were prepared using large pools of purchased human, rat, and mouse sera The first sample in the calibration set was

equivalent to 100% human, mouse, or rat serum (no dilution matrix) and then a series of

22 dilutions were made at a fixed ratio into rabbit or horse serum, respectively Each calibration sample was then mixed 1:4 with PBS to maintain the same fixed dilution in PBS as the experimental samples In the human and mouse calibration sets, two samples were prepared using a higher volume of total serum in PBS (without matrix) than the 100% serum-equivalent samples, to create 125% and 156% of human and mouse serum-equivalents These two samples did not have the same fixed dilution factor into PBS as

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the rest of the calibration samples The total volume of human or mouse serum was larger than the total volume of serum in the rest of the calibration samples Every other calibration sample (12 total) in the dilution series was used to make up one calibration set, called mouse calibration A (Cal-A) and human Cal-G, and the remaining 12 samples created another calibration set, called mouse validation B (Val-B) and human Val-H The rat serum calibration samples were prepared in the same manner, but without

preparation of samples that contained greater-than-100% rat serum The rat serum calibration sets were rat Cal-A and Val-B

Serial dilutions of synthetic apoM tryptic peptides were used as a calibration standard to measure the molar concentration of apoM in these human, mouse, and rat serum calibration samples The molar concentration of apoM in the serum calibration sets was then used as a standard for absolute quantification of apoM in experimental samples One or both sets of species-specific calibration samples were included in each 96-well plate of experimental samples A calibration set was added to the experimental plate at the beginning of sample preparation to undergo the same preparation as the experimental samples prior to MS analysis

LC-MS/ MS of ApoM-Derived Tryptic Peptides

The apoM-derived tryptic peptides used for quantification of apoM include: one peptide common between rat, mouse, and human species (FLLYNR), one human-unique peptide (AFLLTPR), and one rat and mouse-unique peptide (AFLVTPR) The amino acid sequence of apoM was searched against a protein database using Basic Local

Alignment Search Tool (BLAST) from the National Center for Biotechnology

Information (NCBI) [21] to confirm that each tryptic peptide (FLLYNR, AFLLTPR, and AFLVTPR) was unique to apoM A tryptic peptide from the 15N-Apo A-IV

(LEPYADQLR) was used for normalization All of these peptides were measured in a single HPLC gradient and MS method, making this a versatile assay that spans multiple species

The HPLC gradient was optimized using a Surveyor MS pump equipped with an XBridge C18 (2.5 µm particle size) 2.1 mm x 50 mm reverse phase column (Waters, cat

#186003085) The column was kept at 50°C and sample tray was 4°C during the

instrument run 50 µL of 500 µL total sample volume (1 µL of serum) was injected into

a 100 µL sample loop using a partial loop method The HPLC gradient runs 7.5 minutes

per sample and includes the mass-to-charge (m/z) measurement of four tryptic peptides as

2H+-charged precursor ions: FLLYNR (413.50), AFLVTPR (402.49), AFLLTPR

(409.51), and LEPYADQLR (559.58) in a single MS method The HPLC gradient utilized two buffers [0.1% formic acid in H2O (Buffer A) and 0.1% formic acid in

acetonitrile (Buffer B)] The final gradient was: 100% A at 250 µL/min for 0.5 min, 100% A at 300 µL/min for 0.1 min, 0-16% B at 300 µL/min for 0.4 min, 16% B at 300 µL/min for another 4.7 min, 16-80% B at 300 µL/min for 0.3 min, 80% B at 300 µL/min

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for 0.01 min, 80% B at 600 µL/min for 0.49 min, 80-0% B at 600 µL/min for 0.1 min, 100% A at 600 µL/min for 0.9 min, then 100% A at 250 µL/min for 0.1 min The 15N-Apo A-IV-derived tryptic peptide LEPYADQLR eluted around 3.15 minutes and apoM-derived peptides AFLVTPR eluted around 3.89 minutes, FLLYNR close to 4.35 minutes, and AFLLTPR eluted around 5.15 minutes The total run time per injection was 8.5 min including a 1-minute injection, so 170 samples can be analyzed per day Samples were analyzed in tandem to keep HPLC column conditions consistent for all samples The sample was diverted away from the MS source during the first 2.5 minutes of the

analysis The column effluent was also diverted away from the instrument after 5.8 min

to avoid spraying NP40 into the MS source, which was eluted from the column around 6.0 min under this gradient condition

Ion spectra were collected in positive ion mode using an electrospray ionization source (ESI) and a LTQ mass spectrometer The ion capillary temperature was kept at 250°C The maximum ion trap time was set to 25.00 ms The isolation width used to

collect the isotopic distributions of the fragmented tryptic peptides was m/z of 3.00 The

normalized collision energy was 35.00, activation Q was 0.250, and activation time was

25 ms for the MS method Three MS transitions were measured for each apoM-derived tryptic peptide and two for the internal standard-derived tryptic peptide, listed below as:

tryptic peptide, m/z of precursor ion, m/z of transition (fragmentation site, ion charge) AFLLTPR was from m/z of 409.51 to m/z of 599.39 (y5, M+H+ ion), m/z of 486.30 (y4,

M+H+ ion), and m/z of 373.22 (y3, M+H+ ion) FLLYNR was from m/z of 413.50 to m/z

of 565.31 (y4, M+H+ ion), m/z of 452.23 (y3, M+H+ ion), and m/z of 678.39 (y5, M+H+ion) AFLVTPR was from m/z 402.49 to m/z of 585.37 (y5, M+H+ ion), m/z of 472.29

(y4, M+H+ ion), and m/z of 373.22 (y3, M+H+ ion) All tryptic peptides derived from apoM and transitions had an isolation width of 3.00 The internal standard tryptic peptide

LEPYADQLR was from m/z of 559.58 to m/z of 437.19 (y7, M+2H+ ion) and m/z of

873.37 (y7, M+H+ ion) with isolation widths of 3.00 and 4.00, respectively

Tryptic Peptide Peak Identification, Integration, and Quantification

The ion spectra were collected from the fragmentation of each apoM derived tryptic peptide in the targeted assay and were used for peptide identification and apoM quantification Integration of the chromatographic peak of each tryptic peptide was performed using Xcalibur Processing method The chromatographic peak of each

fragment ion was integrated independently from the other two fragments from each precursor ion and the area of each fragment ion chromatographic peak was added

together with the areas of the other two fragment ions from the same peptide prior to curve fitting The peak detection algorithm used in the processing method was

Interactive Chemical Information System (ICIS) [22] ICIS peak integration was set to 3 smoothing points, area noise factor of 1, and a peak noise factor of 10 Peak width was constrained to 10% of the peak height with a tailing factor of 3 The baseline window was adjusted to ensure each peak was fully integrated for consistent quantification The fragment ion with the strongest MS signal (transition A) was detected as the highest peak, and the subsequent fragment ions (transitions B, C) were detected using the nearest retention time based on transition A The minimum peak height of signal to noise ratio was set at 3.0 Noise was filtered out using the repetitive noise function Mass range for

integration was selected for each transition using a narrower window than the m/z

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collection width used in the MS method Transition A from FLLYNR peptide was

integrated using an integration window of m/z 564.81→566.81 amu (M+H+) transition B

between m/z 451.73→453.73 amu (M+H+) and transition C at 677.89→680.89 amu (M+H+) AFLLTPR transition A was integrated at m/z 598.89→600.89 amu (M+H+),

transition B at m/z 485.80→487.80 amu (M+H+), and transition C at m/z 372.72→374.72

amu (M+H+) The mouse peptide, AFLVTPR, had integration windows between m/z

584.87→586.87 amu (M+H+), m/z 471.79→473.79 amu (M+H+), and m/z

372.72→374.72 amu (M+H+) The internal standard tryptic peptide LEPYADQLR

transition A had an integration window of m/z 436.69→438.69 amu (M+2H+), and

transition B at m/z 872.87→874.87 amu (M+H+) The chromatographic peak of each transition was integrated to obtain the area-under-the-curve (AUC) The apoM-derived tryptic peptide AUCs were normalized within each sample as a ratio with the 15N-Apo A-IV-derived tryptic peptide AUC GraphPad Prism software (GraphPad Software, Inc.) was used to fit the calibration sample data to a standard curve and to quantify apoM in the experimental samples using the standard curve A nonlinear regression was used to interpolate the unknown concentrations from the standard curve on a log scale The standard curve was fit as a sigmoidal dose response with variable slope The fitting method was set using the least squares regression and a weighting factor of 1/Y was also used to fit the data

ApoM concentration was reported in relative or absolute concentration In recovery experiments used for method validation, human apoM concentration in Val-H was measured using the known human apoM concentrations in Cal-G (see Results) A three-day spike recovery assay was performed to evaluate the consistency between

spike-triplicates of Cal-G and Val-H (inter-assay) and preparations across three separate

experiments (intra-assay) The coefficient of variation (% CV) was calculated between duplicate plates within an experiment and across three separate experiments to assess the behavior of the assay and its accuracy, precision, and consistency Multiple sets of calibration and validation samples were used for spike recovery The standard deviation and % CV were derived from the comparison of measured apoM concentration against

the known apoM concentrations in the validation standard samples

Adaptation of the Assay to a High-Throughput Format

This assay was optimized for a 96-well plate format to increase throughput The use of a Beckman Coulter Biomek FXP Laboratory Automation Workstation equipped with a Dual Arm system (Span-8 and Multi-Channel Pipettor) (Beckman Coulter, cat

#A31844) robot was incorporated into parts of the sample preparation procedure to further increase throughput and eliminate manual pipetting of samples and reagents to reduce the incidence of sample to sample variations that may occur with multiple manual pipetting steps Preparation of the serum samples was performed entirely in 96-well Captiva filter plates (Varian, cat #A5960045) The bottom of the filter plate was capped with a Captiva Duo Seal plate mat (Varian, cat #A8961008) and fit into a wide mouth 96-well plate (Corning, cat #3433) to keep solution from leaking through the filter plate The Biomek FXP robot delivered 300 µL (250 ng) of iSTD to each well Rabbit serum pre-mixed with PBS (1:6) was used as a background matrix for the serum samples and calibration standards The robot transferred 10 µL of serum samples into 140 µL of dilution matrix to dilute the experimental serum samples 1:2 (3x dilution) with the serum

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matrix Samples were diluted to bring the apoM concentration within the range of the standard curve produced by the calibration samples A 3x dilution brings control serum samples (100% human serum) to 33% At this point on the calibration curve there is approximately a 3-fold range above and below the concentration of apoM in the diluted serum Typically, the experimental samples fell within the calibration range at this dilution factor The robot delivered 50 µL of either the diluted samples or calibration

standards to the filter plate containing the internal standard solution

Liposorb stock solution was added to the filter plates at 200 µL per 10 µL of total serum (sample plus matrix) The tops of the filter plates were sealed with adhesive aluminum foil (Beckman Coulter, cat #538619) Each plate (including bottom cap mat and holder plate) was taped with duct tape in an A to H direction on each end of the filter plate to prevent the filter plates from coming loose at the bottom and leaking during shaking The plates were incubated at 4°C for 30 minutes with shaking to keep Liposorb suspended During this incubation period, the Liposorb beads selectively bound the lipoproteins, purifying them from the complex serum sample in one step This eliminated the need to incorporate an additional high-abundant protein removal step in this protocol The Liposorb fraction contained the apolipoproteins and the unbound fraction was

washed through the filter plate

After incubation with Liposorb, the duct tape and bottom cap mat with holder plate were all removed and the filter plates were placed upright on top of deep 96-well plates The unbound proteins in the supernatant were filtered through the plate, using a centrifuge at 1500 rpm for five minutes to spin down the liquid and collect it in the deep-well plate Operation of the centrifuge at a higher speed resulted in a tight Liposorb

pellet that made resuspension more difficult and possibly incomplete Lower speeds left

a loose pellet to fully resuspend for a more even exposure to trypsin for a consistent digestion The Liposorb pellet with bound apolipoproteins remained in the filter plate and the flow-through was discarded The pellet was washed once with 500 µL of 100

mM ABC and liquid was removed and discarded as before The

apolipoprotein-containing Liposorb pellet was resuspended in either 8 M urea or 100 mM ABC using a Biomek FXP robot

The sample prep protocol diverges at this point into either (1) urea or (2)

reduction and alkylation (R/A) In the urea-containing protocol, the bottom cap mats and holder plate were replaced and samples were resuspended in 100 µL of 8 M urea The filter plate was sealed with adhesive foil and wrapped in duct tape Plates were shaken for 15 minutes at room temperature to keep Liposorb suspended Shaking speeds varied

by plate shaker, so the shaking speed was adjusted per apparatus so that the

apolipoprotein-containing Liposorb beads remained in suspension, but the sample

solution did not come into contact with the adhesive foil to avoid cross-contamination between wells Modified trypsin was added at 2 µg per 10 µL total serum in a final concentration of 0.1% NP40 in 100 mM ABC (pH 8) The final sample was in 500 µL to dilute the final urea concentration to 1.6 M prior to digestion with trypsin Adhesive aluminum foil seals and duct tape were used to keep each filter plate from leaking during digestion At this step it was important to make sure the samples did not reach the

adhesive foil The detergent in the sample can cause the adhesive foil top to unseal and leak Plates were incubated overnight at 37°C, shaking to keep Liposorb suspended to ensure even digestion After digestion, top and bottom seals were removed and samples

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were filtered A filter plate ‘sandwich’ was assembled by placing the bottom of the Varian filter plate into the wells of an additional Solvinert 96-well filter plate (Millipore, cat #MSRPN0410) This assembly was placed on the top of a 96-well plate (Analytical Services Inc, cat #96965) to collect the final digested samples The 96-well plates

containing the final samples were heat-sealed using a non-adhesive foil (AbGene, cat

#AB-0757) compatible with the Surveyor autosampler (Thermo)

In the R/A preparation protocol, the Liposorb pellet was resuspended in 100 µL

10 mM ABC using the Biomek FX Due to the presence of the Liposorb beads, a volatile reduction and alkylation protocol was used to avoid a drying step 10 mM DTT was freshly prepared in 10 mM ABC and added to the suspended Liposorb using a

non-MultiDrop (Thermo) liquid dispenser Plates were sealed with adhesive foil and

incubated at 37°C for 45 minutes with shaking 60 mM Iodoacetamide in 10 mM ABC was then added to the filter plate using the MultiDrop dispenser Plates were incubated at room temperature for 30 minutes in the dark since iodoacetamide is light-sensitive Trypsin was added at the same concentration and solution as the urea protocol (2 µg trypsin per 10 µL total serum) in a final concentration of 0.1% NP40 in 100 mM ABC The final volume in this protocol was also 500 µL Proteins were digested for 2 hours at 37°C in this protocol Post-digestion samples were filtered in the high-throughput format described above 50 µL of 500 µL final volume (1 µL of serum) was injected to the LTQ for measurement of apoM using the MRM assay

Human Clinical Study Samples for Human ApoM Measurement

The conditions of the human serum samples used in this experiment have been described earlier [23] Briefly, serum samples were collected from participants in a 16-week clinical study Each participant was treated with placebo, statin (atorvastatin), PPAR-α agonist (LY518674), or with combination treatments at different doses Human serum samples were prepared using the urea-containing sample preparation protocol and analyzed for apoM-derived tryptic peptide FLLYNR using the MRM assay ApoM was quantified using GraphPad Prism statistical software One-way ANOVA was used to determine statistical difference in group means with p<0.05 Serum was drawn at

baseline (pre-treatment) and at four and 16 weeks post-treatment Measurement of triglycerides, LDL cholesterol, HDL cholesterol, and total cholesterol (mg/ dL) were made and have been described previously [23]

MTTP-Inhibitor Pre-clinical Study for Mouse ApoM Measurement

Mouse apoM was measured in serum samples drawn from mice receiving

different doses of a microsomal triglyceride transfer protein (MTTP) inhibitor or control Mouse serum was prepared using the urea-containing sample preparation protocol and analyzed by MS to measure apoM-derived tryptic peptide AFLVTPR Mouse

apolipoproteins A1, B and E (apoAI, apoB, and apoE) were also measured in these samples using a modified LC-MS method to include measurement of tryptic peptides from additional apolipoproteins in a panel assay HDL, LDL, VLDL, and total

cholesterol were measured independently from this assay

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Sample Preparation for SDS-PAGE and Western Blot of Human ApoM

Human serum and human recombinant apoM were prepared for SDS-PAGE and Western blot to evaluate the use of human recombinant apoM as a standard to quantify apoM in human serum Two sets of dilutions of the recombinant protein were made at

100 µg/mL, 20 µg/mL, and 4 µg/mL, one using PBS as the dilution matrix and the other using 12.5 mg/mL bovine serum albumin, BSA (Pierce, cat #77171) 10 µL of the

diluted recombinant apoM, neat human serum, and BSA was incubated with 200 µL of Liposorb to bind to the apolipoproteins from serum and human recombinant apoM Liposorb supernatant from human serum and all Liposorb fractions were saved and diluted to the same final volume prior to loading on the gel All samples were prepared

in NU PAGE SDS sample buffer (Invitrogen, cat #NP0007) containing NU PAGE

reducing agent (Invitrogen, cat #NP0009) at the same final volume Samples were boiled

at 70°C for 10 minutes Samples were mixed thoroughly and spun down to collect all droplets 0.3 µL of total human serum-equivalent and 1 µg, 0.2 µg, and 0.04 µg of

recombinant apoM were loaded onto a NU PAGE 4-12% pre-cast 1.5 mm Bis-Tris gel (Invitrogen, cat #NP0336BOX) A pre-stained molecular weight standard See Blue Plus2 (Invitrogen, cat #LC5925) was also loaded NU PAGE MES-SDS running buffer was used (Invitrogen, cat #NP0002) with antioxidant (cat #NP0005) included due to reduced sample conditions The samples were run through the gel under reducing conditions according to the Invitrogen NU PAGE Novex Bis-Tris Mini Gels protocol The gel ran at

a constant 200 V for 35 minutes The proteins were transferred to nitrocellulose using the iBlot system from Invitrogen The nitrocellulose was blocked with casein-TBS blocking buffer (Pierce, cat #37532) overnight at 4°C The primary mouse anti-apoM antibody

was added to the nitrocellulose at a 250x dilution in casein TBS buffer and 0.05% Tween

20 detergent (Pierce, cat #PI-28320) The nitrocellulose was washed in 0.05% Tween 20

in TBS The secondary antibody, ECC goat anti-mouse IgG-HRP, was prepared at a 5000x dilution in the same solution as the primary antibody The secondary antibody was removed and washed prior to exposure and film development An ECL Western blot kit (Amersham, cat #RPN2106V1/2) was used for chemiluminescent detection The ECL reagents were mixed 1:1 and added to the nitrocellulose for one minute Kodak BioMax Light film was exposed to the nitrocellulose at different time intervals and developed

In an experiment to evaluate the selective nature of Liposorb for apolipoproteins from serum, aliquots of 10 µL of human serum and rat plasma (in-house Long-Evans) were incubated with 200 µL of Liposorb and analyzed along with neat human and rat serum by SDS-PAGE The gel was stained with Invitrogen Simply Blue SafeStain (cat

#LC6060) and washed with H2O overnight The visible protein bands were excised, destained with 50% ACN in 10 mM ABC and digested with modified trypsin The gel slices were removed from the digested sample by filtration The peptides from each gel slice were analyzed by nanospray LC-MS/ MS (LTQ) in a global profiling experiment using a triple-play data collection method and searched against human and rat protein databases for protein identification

In another experiment, apoM concentration was measured in several human serum samples using the MS-based assay and compared to the apoM concentration measurements from a more conventional proteomic method, Western blot, in an

orthogonal validation of the MS assay Aliquots of the same human serum samples containing different concentrations of apoM were analyzed using both methods The MS

24

measurement was performed using the urea-containing method as described above Serum samples were also prepared for SDS-PAGE Five microliters (5 µL) of human serum was diluted into 27.5 µL H2O, 5 µL of 10x NU PAGE reducing agent, and 12.5 µL

of 4x NU PAGE SDS sample buffer to a total volume of 50 µL Ten microliters (10 µL)

of each sample (1 µL serum equivalent) was loaded onto a NU PAGE 4-12% pre-cast 1.5

mm Bis-Tris gel 10 µL of SeeBlue Plus2 pre-stained MW standard was loaded into the first well Serum samples (numbered 1-12) were loaded in order into wells 2-13 Well

14 contained 10 µL of the MW standard and well 15 contained the same sample as well

10 (sample 9) A negative control for primary antibody was included in this blotting procedure Mouse anti-apoM primary antibody was incubated with a 10x excess of recombinant apoM before incubation with a selected portion of the blot (well 15) The remaining portion of the blot was probed with the primary antibody without the presence

of recombinant apoM, as usual The two sections were then placed together during the secondary antibody probe and ECL exposure The SDS-PAGE was run according to the

NU PAGE Novex Bis-Tris Mini Gels protocol from Invitrogen (described above) The proteins in the gel were transferred to nitrocellulose using the iBlot system from

Invitrogen The nitrocellulose was kept in Pierce Superblock in TBS (cat #37535)

overnight at 4°C to block non-specific antibody binding sites The nitrocellulose

membrane was incubated for one hour with primary antibody mouse anti-apoM diluted 250x in casein-TBS with 0.05% Tween 20 The blots were washed and then incubated for 45 minutes with anti-mouse IgG secondary antibody The blots were washed a

second round Amersham ECL Western blot kit was used to expose and develop the Western blot as described above

RESULTS

Enrichment of Apolipoproteins from Serum using Liposorb

Liposorb is known to capture lipids and lipid-binding proteins from serum or plasma and has been used previously to quantitatively capture apolipoproteins [20] We tested the selective nature of Liposorb for apolipoproteins and also tested if this

procedure can be used to quantitatively capture apoM and applied to other

apolipoproteins as well The selective nature of Liposorb was tested by SDS-PAGE comparison of Liposorb-bound human serum and neat human serum to confirm that apolipoproteins were the primary proteins identified from the Liposorb-bound fraction apoB100, apoAI, apoE, apoAIV, apoCIII, and apoAII were the major proteins identified

by global MS analysis of the resulting protein bands from the Liposorb-bound fraction of human serum (Figure 2) A few other lipid-binding proteins were also identified in these bands, but the Liposorb primarily bound apolipoproteins from serum The Liposorb-bound fraction contained visibly less protein bands than the neat serum when 1 µL of human serum-equivalent was loaded for each condition and directly compared The protein bands in the Liposorb-bound fraction of 1 µL human serum were not intense enough to be excised and digested for MS preparation and analysis, so 10 µL of

Liposorb-bound human serum-equivalent was also loaded and this lane was used for protein identification by global proteomics using MS ApoM was not identified in any of the protein bands from this experiment, likely due to its low abundance which can result

in a very light or absent protein band The results of this experiment confirmed the selective nature of Liposorb for removal of apolipoproteins from serum and can be

26

applied to other apolipoprotein assays, but did not address the selective or quantitative capture of apoM by Liposorb for use in this assay

Quantitative capture of apoM by Liposorb was evaluated using Western blot

Human serum and recombinant human apoM purified from E.coli were incubated with

Liposorb and both Liposorb-bound and unbound supernatant fractions were analyzed for apoM by Western blot An apoM band was not visible in the supernatant of either sample, but both Liposorb-bound recombinant and human serum had an intense apoM band around 25 kDa (Figure 3) The intense presence of apoM in the Liposorb-bound fraction, in conjunction with its absence in the supernatant, demonstrated the selective and quantitative capture of apoM from serum by Liposorb Apolipoproteins were

selectively removed from serum and apoM was quantitatively captured by Liposorb ApoM protein band intensity from Liposorb-bound apoM from human serum was not compared to the band intensity of neat human serum in this analysis, but quantitative capture of apoM was later confirmed using MS analysis

Thus, Liposorb was included in sample preparation prior to MS analysis Serum was incubated with Liposorb to selectively capture apolipoproteins The sample was spun down to pellet the Liposorb, the supernatant containing unbound proteins was removed, and the Liposorb pellet was washed prior to trypsin digestion and injection into the MS for analysis Liposorb treatment was also be used prior to Western blot analysis

or other proteomic method in which selective analysis of apolipoproteins was desired

Figure 2

188 98

62 49 38 28 17 14 6

3

188 98

62 49 38 28 17 14 6

3

188 98

62 49 38 28 17 14 6

3

10 µL 1 µL 1 µL MW kDa

Human Serum + Liposorb

Human Serum

ApoB-100

Serotransferrin

ApoL-1, ApoA-1

ApoA-1 PON-1, ApoA-4

ApoE, ApoL-1, PON-1

ApoE, ApoA-1, ApoL-1

ApoA-1 ApoA-1, ApoE

Amyloid A-4 Amyloid A/A-4

ApoC-3 ApoA-2, ApoA-1

188 98

62 49 38 28 17 14 6

3

188 98

62 49 38 28 17 14 6

3

188 98

62 49 38 28 17 14 6

3

10 µL 1 µL 1 µL MW kDa

Human Serum + Liposorb

Human Serum

ApoB-100

Serotransferrin

ApoL-1, ApoA-1

ApoA-1 PON-1, ApoA-4

ApoE, ApoL-1, PON-1

ApoE, ApoA-1, ApoL-1

ApoA-1 ApoA-1, ApoE

Amyloid A-4 Amyloid A/A-4

ApoC-3 ApoA-2, ApoA-1

Figure 2: Selective capture of apolipoproteins from human serum using Liposorb

SDS-PAGE demonstrates the selective capture of apolipoproteins and other

lipid-associated proteins from human serum using PHM-Liposorb (rat serum SDS-PAGE not shown) The indicated gel bands were excised from the gel, digested, and proteins were identified using nanospray LC-MS/ MS The major proteins in each band were mostly identified as apolipoproteins and are listed next to the corresponding arrows Protein bands were analyzed from 10 µL of Liposorb-bound human serum and the major protein identifications are shown above 1 µL of Liposorb-bound human serum was compared to

1 µL of neat human serum and was shown that the Liposorb-bound fraction has lower protein content than the whole serum These bands were identified as mostly higher abundant apolipoproteins, demonstrating the selectivity of Liposorb to capture

apolipoproteins ApoA-I was identified in several bands, possibly due to proteolytic

Hm Serum

-36 -22

Hm Serum

-36 -22 MW

Figure 3: Quantitative capture of apoM from human serum and recombinant

protein using Liposorb

The quantitative capture of apoM was evaluated using the results of Western blot

analysis Human serum was incubated with Liposorb to selectively bind apolipoproteins The supernatant was then removed and both fractions were prepared for SDS-PAGE and Western blot analysis of apoM ApoM from human serum and human recombinant apoM were both captured by Liposorb with no detectable apoM remaining in the supernatant The quantitative capture of apoM was demonstrated using Western blot and later

confirmed with MS analysis

Detergent with Trypsin Digestion to Increase Recovery of Some Apolipoproteins

A MS-based assay for quantification of multiple apolipoproteins was developed in conjunction with this specific apoM assay A few of the apolipoprotein B (apoB) and paraoxonase-1 (PON1)-derived tryptic peptides had a low recovery from human serum and their synthetic tryptic peptides were insoluble An additional step in sample

preparation was needed to keep select synthetic peptides in solution throughout

preparation and during MS analysis and increase the recovery of these apolipoproteins from serum

Detergent was evaluated for potential use in this assay to increase protein and peptide solubility and recovery from serum, although detergent is not typically used in

MS preparation due to its high ionization efficiency and dominating MS spectra Several different detergents and concentrations were evaluated to determine the lowest

concentration of detergent that yielded the highest recovery Pierce Surfact-Amps NP40 and Surfact-Amps X100 (cat #28314) were evaluated at 0.01%, 0.05%, and 0.1% using the synthetic peptides of each apolipoprotein in the panel assay (unpublished assay) These peptides were synthesized as tryptic peptides, so detergent was diluted into 100

mM ABC and added directly at these final concentrations Synthetic peptides were analyzed using the targeted MS apolipoprotein panel assay and recovery was calculated

as a percentage of control (no detergent) Detergent was also used to increase protein recovery from serum Human serum was prepared using the method described above without detergent (control) and with the addition of detergent prior to trypsin digestion NP40 and Triton X100 were evaluated at 0.01%, 0.05%, and 0.1% and Waters RapiGest

SF Surfactant (cat #186001860) was used at 0.001%, 0.005%, and 0.01%

30

The addition of TritonX100 or NP40 at any percentage prior to MS analysis was found to increase the peptide MS signal of apoB synthetic peptides more than 100-fold and PON-1 synthetic peptides more than 4-fold without detrimental effects to the MS signal of the other synthetic peptides in the panel of apolipoproteins (Figure 4A) The addition of 0.1% NP40 prior to digestion with trypsin increased the recovery of apoB from human serum by 10-fold and did not decrease the recovery of others (Figure 4B) PON-1 was increased 5-fold with the addition of 0.001% RapiGest, but this surfactant should be neutralized with acid prior to MS analysis and did not increase the recovery of apoB, so was not used The final selection was 0.1% NP40 added during trypsin

digestion of serum and was included in synthetic peptide preparations to keep the

peptides soluble, and use of one detergent and the same concentration keeps the

preparation procedure simplified Detergent is not commonly used in sample preparation prior to MS analysis, but since this is a targeted method, the detergent was eluted from the HPLC column at a high concentration of acetonitrile, after all of the target peptides were eluted and measured, and the detergent spray was diverted away from the MS source to the waste This helped maintain a cleaner instrument and did not interfere with the measurement of the target tryptic peptides in this method

ApoM was not included in these experiments because they were performed earlier

in method development of other apolipoproteins and prior to the development of this assay, but the affect of detergent on apoM recovery was evaluated during the

optimization of the apoM assay using the targeted MS assay and is later described in detail The use of 0.1% NP40 during trypsin digestion was thus included in sample

preparation for apoM analysis to maintain consistency with the panel assay until further experiments were performed

Detergent Increases ApoB and PON-1-Derived

Peptide Recovery from Human Serum

Detergent Increases ApoB and PON-1-Derived

Peptide Recovery from Human Serum

Figure 4: Evaluation of different concentrations of different detergents to increase apolipoprotein B and paraoxonase-1 recovery and solubility

Triton X100 (TX), NP40 (NP), and RapiGest (RG) were investigated to increase the solubility of apolipoprotein B (apoB) and paraoxonase-1 (PON1) synthetic peptides and apoB and PON-1 protein recovery from human serum The control condition (ctrl) did not include detergent during trypsin digestion

(4A) Different concentrations of detergent were added to synthetic peptides prior to MS analysis to increase solubility, reflected in an increase in synthetic peptide MS signal when compared against the detergent-free synthetic peptide MS signal, indicating an increase in solubility

(4B) Different detergents and RapiGest were evaluated to increase apoB and derived tryptic peptide recovery from human serum The addition of 0.1% NP40 prior to digestion with trypsin resulted in the highest recovery of apoB peptides from human serum compared to control This concentration of NP40 did not suppress the recovery of other apolipoprotein-derived tryptic peptides and was thus included in apolipoprotein sample preparation during trypsin digestion 0.1% NP40 was also included during trypsin digestion in the sample preparation protocol for the targeted apoM assay

PON-1-34

Identification of ApoM-Derived Tryptic Peptides using Global Proteomics

Unbiased global proteomics studies were initially used to identify apoM in human and rat serum and to guide selection of tryptic peptides for development of a Multiple Reaction Monitoring (MRM) method Different aliquots of human and rat serum were treated with Liposorb and digested with trypsin prior to MS analysis These samples were analyzed by LC-MS/ MS using a triple-play global proteomic method to identify unique peptides derived from Liposorb-bound proteins Triple-play LC-MS/ MS

performs three scans per peptide: full MS scan, zoom scan, and MS/ MS scan The most abundant peptide present in the full MS scan was selected for zoom scan and then

fragmented prior to full MS/ MS scan The zoom scan estimated the charge state of the peptide and the full MS/ MS scan collected spectra from the product ions generated in the fragmentation of the precursor ion (tryptic peptide) The full MS/ MS spectra was used

in database searches to obtain protein identifications (Experimental Section) and manual analysis of the apoM-derived tryptic peptide fragmentation spectra was used to build the targeted MS assay

Three unique human apoM-derived tryptic peptides were identified from the global studies: AFLLTPR, WIYHLTEGSTDLR, and

EELATFDPVDNIVFNMAAGSAPMQHLR Rat serum analysis also produced three unique apoM-derived tryptic peptides: KWTYHLTEGK, AFLVTPR, and FLLYNR Several criteria were used to evaluate whether or not these tryptic peptides are suitable to include in the targeted assay, i.e the presence of potential modification sites such as the potential of methionine to be oxidized [19]

Elution of the tryptic peptides from the HPLC column by acetonitrile was

recorded in the global study as retention time (RT) Human apoM-derived tryptic

peptides AFLLTPR, WIYHLTEGSTDLR, and

EELATFDPVDNIVFNMAAGSAPMQHLR had retention times of 34.7, 39.6, and 74.5 minutes on the 142-minute gradient, respectively Rat apoM-derived tryptic peptides KWTYHLTEGK, AFLVTPR, and FLLYNR had retention times of 21.5, 28.6, and 33.2 minutes, respectively The retention time of each apoM-derived tryptic peptide was used

to estimate the percent of acetonitrile (ACN) needed to elute the peptides from the

reverse-phase HPLC column based on the 142-minute gradient (Table 1) to facilitate development of a shortened gradient for the targeted human and rat apoM methods To keep the HPLC gradient for the targeted method as short and simple as possible, the use

of tryptic peptides that were extremely hydrophilic or hydrophobic based on the retention time in the global studies was avoided

To develop a quantitative assay, it was important to avoid the selection of tryptic peptides that contained potential modifications sites Peptide modifications such as methionine oxidation cause a shift in mass compared to the mass of the unmodified peptide When this occurs, the modified peptide will have a different mass-to-charge

ratio (m/z) than the unmodified peptide m/z, which is collected by the targeted method

and so the unmodified peptide spectra will not be collected Due to the presence of methionine in EELATFDPVDNIVFNMAAGSAPMQHLR, this human apoM-derived tryptic peptide was not included in the targeted method, since methionine can be readily oxidized Thus, quantification of apoM was not based on a tryptic peptide that may become modified, to provide an accurate measurement of apoM The other apoM-

The tryptic peptides identified from apoM in the global studies were evaluated for their presence at multiple charge states Human tryptic peptide WIYHLTEGSTDLR and rat tryptic peptide KWTYHLTEGK were identified at both 2H+ and 3H+ charge states (Table 2) Human peptide AFLLTPR and rat peptides FLLYNR and AFLVTPR were found at one charge state (2H+)

Although these two tryptic peptides were each identified at two different charge states, they were later evaluated using preliminary MRM studies for the consistency of the ratio of these two charge states and peptide MS signal intensity Two human

(AFLLTPR and WIYHLTEGSTDLR) and three rat apoM-derived tryptic peptides

(AFLVTPR, FLLYNR, and KWTYHLTEGK) were all included in further evaluation for

use in the MRM assay

Table 1: ApoM-derived tryptic peptides identified in global studies

The initial results from global profiling studies identified three human apoM-derived tryptic peptides (top) and three rat apoM-derived tryptic peptides (bottom) The triple-play MS method identified each unique tryptic peptide and the retention time (RT) based

on the 142-minute HPLC gradient was captured Retention time from the global studies was used to estimate the percent of acetonitrile needed to elute each peptide from the HPLC column to estimate a starting gradient for the initial targeted method setup for human and rat apoM methods

Full MS/ MS Spectra of ApoM-Derived Tryptic Peptides Identified in Global

Studies

Full MS/ MS spectra of each tryptic peptide was collected from global studies and used to select the three most abundant fragments from each tryptic peptide The tryptic peptide and its three fragment ions were needed to set up the MRM assay Each tryptic peptide produces its unique fragmentation pattern that can be entered into the MRM method and used for identification and quantification of the target protein (apoM) This

method is set up using the m/z ratio of the tryptic peptide (precursor ion) and m/z ratios of

three fragment ions per tryptic peptide In the targeted MRM method, the MS scans for

these m/z values only

The spectra collected in the full MS/ MS scan of the fragmented apoM-derived tryptic peptides (Figure 5) were manually evaluated to select the most abundant fragment

ions per tryptic peptide Each tryptic peptide (m/z of the precursor ion) and three

fragment selections (m/z of each fragment) were: human AFLLTPR peptide was from m/z

of 409.51 to m/z of 599.39 (y5, M+H+ ion), m/z of 486.30 (y4, M+H+ ion), and m/z of

373.22 (y3, M+H+ ion); human WIYHLTEGSTDLR peptide (3H+) was from m/z of 531.26 to m/z of 646.32 (y11, M+2H+), m/z of 652.30 (b11, M+2H+), and m/z of 472.24

(b7, M+2H+); and human WIYHLTEGSTDLR peptide (2H+) was from m/z of 796.39 to

m/z of 878.42 (y8, M+H+), m/z of 646.32 (y11, M+2H+), and m/z of 991.51 (y9, M+H+)

Rat FLLYNR peptide selections were from m/z of 413.50 to m/z of 565.31 (y4, M+H+ion), m/z of 261.16 (b2, M+H+), and m/z of 452.23 (y3, M+H+ ion); rat AFLVTPR

peptide was from m/z 402.49 to m/z of 585.37 (y5, M+H+ ion), m/z of 472.29 (y4, M+H+ion), and m/z of 373.22 (y3, M+H+ ion); rat KWTYHLTEGK (3H+) was from m/z of

40

421.82 to m/z of 567.78 (y9, M+2H+), m/z of 530.27 (b8, M+2H+), and m/z of 415.22 (b6,

M+2H+); and rat KWTYHLTEGK (2H+) was from m/z of 632.22 to m/z of 622.85 (b10,

M+2H+), m/z of 567.78 (y9, M+2H+), and m/z of 1134.56 (y9, M+H+) and were

summarized in Table 3 These selections were used to set up a preliminary MRM method for further peptide evaluation