>> LET'S GET STARTED.
SO, TODAY IT'S A GREAT PLEASURE
TO WELCOME DR. BRIAN SEARLE TO
OUR PROTEOMICS INTEREST GROUP
SEMINAR.
TO ME, BRIAN IS KIND OF A LEGEND
IN HIS OWN WAY.
YOU'LL FIND OUT.
SO, BRIAN IS ASSISTANT
PROFESSOR, OHIO STATE UNIVERSITY
MEDICAL CENTER, DEPARTMENT OF
BIOMEDICAL INFORMATICS, A MEMBER
OF THE INSTITUTE FOR
IMMUNO-ONCOLOGY, RECEIVED
BACHELOR'S DEGREE IN CHEMISTRY
IN 2001, AND GO DIRECTLY TO GRAD
SCHOOL.
IN 2004, HE CO-FOUNDED THE
PROTEOME SOFTWARE COMPANY WITH
MARK TURNER AND DR. ASHLEY
McCOMAK TO PRODUCE AND
DISTRIBUTE CUTTING EDGE
SOFTWARE.
A LOT OF PEOPLE DON'T ASSOCIATE
PROTEOME SOFTWARE WITH BRAND
NAME SCAFFOLD, AND MOST OF US
DOING PROTEOMICS IN EARLY DAYS,
LATE 2000s, EARLY 2010 YEARS,
KNOW THE SOFTWARE SCAFFOLD.
THAT'S BASICALLY THE GO-TO
SOFTWARE FOR PROTEOMICS,
ESPECIALLY FOR CORE FACILITIES.
THAT'S A SUCCESSFUL BUSINESS AND
WE ALL RELY ON ITS PUBLICATIONS
FOR QUITE A WHILE UNTIL PROTEOME
DISCOVERY CAUGHT UP.
BUT THEN IN 2014, HE DECIDED HE
WENT BACK TO GRADUATE SCHOOL,
AND GOT HIS Ph.D. WITH DR.
MIKE McCASS, UNIVERSITY OF
WASHINGTON, WHERE HE DEVELOPED
METHODS TO DETECT PROTEINS USING
MASS SPECTROMETRY.
SO, OF COURSE THAT'S A
SUCCESSFUL Ph.D. PROGRAM AND
THEN NOW HE'S STARTED HIS OWN
LAB IN 2018?
>> (INAUDIBLE).
>> OKAY.
IN 2018.
AND THEN MOVED TO OHIO STATE IN
2019.
AND HIS LAB SPANS INTERSECTION
OF PROTEOMICS, MASS
SPECTROMETRY, BIOINFORMATICS AND
TECHNOLOGY, DEVELOPMENT TO STUDY
HUMAN GENETIC VARIATION WITH
CANCER.
HERE IS BRIAN, WITHOUT FURTHER
ADO.
>> THANKS.
YEAH, I HAD A SMALL LAB AT ISB.
VERY LIMITED LAB, ONE POSTDOC,
WE WERE DOING MOSTLY
COMPUTATIONAL WORK AT THE TIME.
BUT IT LED INTO THIS LAB THAT
I'VE BEEN BUILDING AT OSU.
SO, TODAY I WANT TO TELL YOU
SOMETHING, A STORY ABOUT
SOMETHING THAT'S DIFFERENT THAN
WHAT I USUALLY TALK ABOUT.
MY BACKGROUND IS COMPUTATIONALLY
ORIENTED.
I STARTED IN A BIOINFORMATICS
POSITION, AND I STARTED A
BIOINFORMATICS COMPANY.
I WENT TO GRAD SCHOOL MOSTLY, IN
PART AT LEAST, FOCUSING ON
BIOINFORMATICS.
MANY OF MY TALKS ARE AROUND
BIOINFORMATICS TYPE OF
QUESTIONS.
THIS IS NOT ONE OF THEM.
THIS IS A PROJECT THAT'S A
BIOLOGY PROJECT.
AND WHAT'S EVEN WEIRDER IS IT'S
A BIOMARKER DISCOVERY PROJECT.
WHEN I STARTED MY LAB, THE FIRST
THING I SAID WAS I'M NEVER GOING
TO TAKE A BIOMARKER DISCOVERY
PROJECT ON.
THAT'S THE LAST THING I WANTED
TO DO.
I WANT A MECHANISM, HYPOTHESIS,
I WANT TO KNOW WHAT I'M
STUDYING, TO MAKE SURE THAT I'M
MAKING THE RIGHT CHOICES FOR
THAT.
SO, THE PROJECT THAT I WANT TO
TALK TO YOU TODAY ABOUT IS ABOUT
IDENTIFYING THESE NOVEL
PANCREATITIS BIOMARKERS
SPECIFICALLY IN URINE SAMPLES.
THIS IS INTEREST FROM MY
PERSPECTIVE, A DEVIATION, SO TO
ME THIS IS A NEW TALK.
WE SHOULD HAVE FUN WITH IT.
IF YOU HAVE QUESTIONS ALONG THE
WAY, I'D PREFER THEM AS WE'RE
GOING.
RAISE YOUR HAND, ASK THEM, IT
WILL BE MORE FUN IF IT'S
INTERACTIVE.
COOL?
OKAY.
SO THE FIRST THING I WANT TO DO
IS TALK ABOUT SOME DISCLOSURES.
SO, AS WAS SAID, I'M EMPLOYED AS
ASSISTANT PROFESSOR AT OHIO
STATE UNIVERSITY.
BUT I'M ALSO THE FOUNDER OF THE
COMPANY SCAFFOLD, I'M ALSO ON
THE SCIENTIFIC ADVISORY BOARD
FOR TALUS BIOSCIENCES.
AND MY RESEARCH IS FUNDED
THROUGH NIGMS AND THROUGH THE
NCI.
THE FIRST THING I WANT TO TALK
ABOUT, AS A CHILD OF THE '80s,
LIKE WE WOULD SEE SLOGANS LIKE
THIS FOR DARE.
FRIENDS DON'T LET FRIENDS DO
DRUGS.
I WANT TO CHANGE THIS AROUND A
LITTLE BIT, BECAUSE WHAT I WANT
TO SAY, FRIENDS DON'T LET
FRIENDS USE ANTIBODIES AS
READOUTS.
SPECIFICALLY, LET ME SEE IF I
CAN MOVE THAT.
NO?
WE'LL GIVE UP ON THAT.
AS AN UNVALIDATED READOUT.
AND THIS TALK IS ABOUT REALIZING
ANTIBODIES AT LEAST FOR CERTAIN
TYPES OF EXPERIMENTS, DON'T
NECESSARILY WORK.
AND THAT A LOT OF MY TALKS MORE
RECENTLY HAVE TURNED INTO THIS
DIAL OF TALK, UNFORTUNATELY,
BECAUSE OF THE PREVALENCE
THROUGHOUT THE COMMUNITY.
SO, THE QUESTION I WANT TO TALK
TO YOU ABOUT TODAY IS ON
PANCREATIC DISEASE.
SO, ACUTE PANCREATITIS IS ONE OF
THE BIGGEST, ONE OF THE MOST
IMPORTANT PANCREATIC DISEASES
FROM THE STANDPOINT OF THE
NUMBER OF PEOPLE IT AFFECTS, AND
THAT ACUTE PANCREATITIS IS ONE
OF THE MOST COMMON G.I.
DISORDERS IN GENERAL.
AND PARTICULARLY IN THAT IT HAS
SORT OF VERY UNUSUAL PHYSIOLOGY,
RIGHT?
IT STARTS AT VERY DIFFERENT AGES
FOR DIFFERENT PEOPLE.
IT HAS VERY DIFFERENT
ASSOCIATIONS WITH WHAT'S GOING
ON.
AND DESPITE SORT OF THE -- LIKE
THESE ISSUES WITH TRYING TO MAKE
MEASUREMENTS AND HOW IMPORTANT
IT IS, IT'S REALLY KIND OF
CHALLENGING TO TRY TO MAKE
MEASUREMENTS OF IT.
SPECIFICALLY BECAUSE THERE ARE A
LOT OF CO-MORBIDITIES ASSOCIATED
WITH PANCREATIC DISEASES IN
GENERAL, ACUTE PANCREATITIS AND
CHRONIC PANCREATITIS.
WE'LL GET TO SOME A LITTLE BIT
LATER.
BUT SPECIFICALLY, PANCREATITIS
HAS BEEN STUDIED IN VERY LARGE
COHORTS SURROUNDING ALCOHOLISM
AND SMOKING, WHICH TEND TO BE
THE BIGGEST DRIVERS FOR
PANCREATIC DISEASE IN GENERAL,
SPECIFICALLY WITH CHRONIC
PANCREATITIS.
SO, FOR ACUTE PANCREATITIS, WE
DO HAVE BIOMARKERS FOR THIS
DISEASE.
AND LIPASE IS THE ONE THAT IS
THE MOST COMMONLY USED.
LIPASE IS A BIOMARKER THAT WAS
DISCOVERED IN THE EARLY 1900s.
AND WAS FOUND TO BE INCREASED IN
ACUTE PANCREATITIS, AS A
POTENTIAL MARKER FOR THE
DIAGNOSIS FOR THE DISEASE.
BUT HONESTLY WE MAY HAVE BEEN
TAKING LIPASE FOR GRANTED AS A
FIELD IN THE USE OF THIS TYPE OF
BIOMARKER, JUST BECAUSE OF OTHER
SOURCES OF ELEVATION OF LIPASE.
FOR EXAMPLE, WE SEE THAT IN THE
PANCREAS, FOR EXAMPLE, THERE'S A
HIGHER LEVEL OF LIPASE JUST SORT
OF GENERALLY, NOT JUST IN -- NOT
JUST IN ACUTE PANCREATITIS OR
CHRONIC PANCREATITIS, BUT AS
WELL THERE ARE REASONABLY HIGH
LEVELS OF LIPASE IN OTHER ORGANS
AS WELL, OTHER BIOFLUIDS AS
WELL.
AS A RESULT, IT IS A COMPLICATED
BIOMARKER TO USE.
BECAUSE FIGURING OUT WHAT ARE
THE APPROPRIATE LEVELS FOR THIS
IN A DISEASE POPULATION CAN BE A
LITTLE BIT CHALLENGING BECAUSE
THE DIFFERENCE BETWEEN WHETHER
SOMEONE HAS THE DISEASE AND
WHETHER THEY DON'T IS VERY
SHALLOW MARGIN OF ERROR FOR
MAKING A CALL, AS TO WHETHER
THEY HAVE -- THAT THEY HAVE A
DISORDER.
SO, LIPASE IS A CHALLENGE FOR A
WIDE VARIETY OF REASONS.
AND IT CAN LEAD TO MISSED
DIAGNOSIS IN LOTS OF DIFFERENT
CASES.
THIS DIAGNOSIS IN THESE CASES
HAS PRETTY MAJOR CONSEQUENCES.
IN FACT, GOING ALL THE WAY TO
SORT OF LIKE PANCREAS REMOVAL,
AND THAT SORT OF THING.
SO LOTS OF DISEASES CAN ACTUALLY
CAUSE INCREASED LIPASE, SO
THERE'S A WIDE VARIETY OF
INTERABDOMINAL DISEASES THAT CAN
BE ASSOCIATED WITH INCREASED
LIPASE.
BUT ALSO IT CAN BE ASSOCIATED
WITH TOTALLY DIFFERENT THINGS AS
WELL.
SO, IT CAN HAVE -- BE RESULTING
IN SORT OF REDUCE THE CLEARANCE
OF LIPASE IN GENERAL.
ASSOCIATED WITH A VARIETY OF
THINGS LIKE BRAIN DAMAGE, FOR
EXAMPLE, OR JUST DRUG USAGE, FOR
EXAMPLE.
AND SO, AS A RESULT, THERE'S
BEEN A MAJOR PUSH BY THE
PANCREAS COMMUNITY TO START TO
LOOK FOR DIFFERENT TYPES OF
APPROACHES TO DIAGNOSING THIS
PARTICULAR DISEASE AND TREATING
THIS DISEASE.
SO IN PARTICULAR, THE TOP GOAL
FOR ACUTE PANCREATITIS RESEARCH
PUBLISHED IN THIS MAJOR SURVEY
WAS TRYING TO FIGURE OUT WAYS TO
TREAT PANCREATITIS WITHOUT USING
KIND OF OPIATE-STYLE DRUGS THAT
WE KNOW ARE QUITE PREVALENT.
SO THE PAIN MANAGEMENT IS ONE OF
THE KEY ASPECTS OF IT.
THIS IS DEFINITELY A DIRECTION
THAT MY LAB HAS BEEN FOCUSING IN
MORE RECENTLY.
BUT JUST TRYING TO FIGURE OUT
WHEN SOMEONE PRESENTS WITH
HAVING ABDOMINAL PAIN SAYING DO
THEY ACTUALLY HAVE PANCREATITIS
AS A POTENTIAL AVENUE FOR DOING
DIAGNOSIS, HAVING QUICK AND
EARLY ACCESS TO GOOD BIOMARKERS
ARE GOOD IDENTIFICATION OF THE
DISEASE, CAN BE REALLY CRITICAL
TO HOW WELL THAT THEY CAN
SUCCEED ON TREATMENT, FOR
EXAMPLE.
SO, THIS IS THE DISEASE THAT HAS
A WIDE VARIETY OF EXISTING
POTENTIAL BIOMARKERS THAT
ALREADY EXIST.
SO, WE TALKED ABOUT LIPASE, SORT
OF THE GOLD STANDARD FOR DOING
DIAGNOSIS, AND IT'S BEEN THE
STANDARD OF CARE FOR ALMOST 100
YEARS AT THIS POINT.
SERUM AMYLASE IS A NEWER MARKER
THAT IS ALSO THE OTHER SORT OF
HALLMARK OF THIS DISEASE.
SO, BEING ABLE TO DETECT SERUM
AMYLASE AT CERTAIN LEVELS TENDS
TO BE REALLY BENEFICIAL FOR
MAKING A DETECTION.
THERE'S ANOTHER EXISTING
BIOMARKER, TRYPSINOGEN,
IDENTIFIED AS USED IN EUROPE
PRACTICE BUT NOT APPROVED IN THE
U.S. AT THIS POINT.
THERE ARE A FEW OTHER
BIOMARKERS.
WE'LL COME BACK TO SOME OF THESE
A LITTLE BIT LATER.
BUT KNOW THAT THERE'S BEEN WORK
IN THIS AREA TO TRY TO IDENTIFY
OTHER MARKERS OF THIS DISEASE,
IN GENERAL THESE ARE NOT BEEN AS
PARTICULARFUL, -- SUCCESSFUL,
IDENTIFIED BUT NOT USED.
I THINK THIS IS AN INTERESTING
PROBLEM, BUT I WOULDN'T GET INTO
THIS TYPE OF THINK AS AN
ANALYTICAL CHEMIST IF IT WEREN'T
FOR SORT OF THE TRYING TO SEE
THERE WAS AN OPPORTUNITY AND
TRYING TO MAKE A MEASUREMENT.
AND THE OPPORTUNITY FOR MAKING A
DIFFERENCE HERE ACTUALLY CAME
FROM THIS COLLEAGUE OF MINE,
MAISAM, FROM CINCINNATI
CHILDREN'S.
MAISAM APPROACHED ME A COUPLE
YEARS AGO AND SON THAT SHE WAS
INTERESTED IN TRYING TO STUDY
THIS PARTICULAR DISEASE, AND
TRIED TO MAKE PROGRESS IN IT.
BUT SHE WAS FOCUSING ON
SOMETHING THAT WAS A LITTLE BIT
DIFFERENT, NOT TRYING TO
IDENTIFY PANCREATITIS IN THE
GLOBAL POPULATION BUT
SPECIFICALLY TRYING TO IDENTIFY
IT IN CHILDREN.
SO, IF YOU THINK BACK, WHAT ARE
THE MOST IMPORTANT
CHARACTERISTICS OR WHAT ARE THE
MOST LIKELY DRIVERS OF
PANCREATIC DISEASE?
THEY ARE GOING TO BE THINGS LIKE
SMOKING AND DRINKING, AND DRUG
USE, OBESITY ARE GOING TO BE THE
BIGGEST DRIVERS THAT HAVE BEEN
ASSOCIATED WITH THE DISEASE
ALREADY.
AND SO ONE ADVANTAGE WITH
WORKING IN A CHILDREN'S
POPULATION IS WE HAVE TO WORRY A
LOT LESS ABOUT THE IMPACT OF
DRUG USE, IMPACT OF DRUGS AND
ALCOHOL, ON THEIR LIKELIHOOD OF
GETTING THE DISEASE, AND WE ALSO
HAVE TO WORRY A LITTLE BIT LESS
ABOUT OBESITY BEING AN ISSUE.
WE DON'T HAVE TO WORRY QUITE SO
MUCH ABOUT AGE BEING AN ISSUE,
BECAUSE THE OLDER YOU ARE, THE
MORE LIKELY YOU'RE GOING TO
PRESENT.
SO, THERE'S SOME REAL
INTERESTING ADVANTAGES TO BEING
ABLE TO WORK WITH THIS DISEASE
IN CHILDREN, IN THAT WE CAN HONE
IN SPECIFICALLY ON MAYBE
POTENTIAL MARKERS THAT ARE
UNIQUE TO THE DISEASE AND NOT
UNIQUE TO THE PHENOTYPE ITSELF.
OR THE OTHER ASSOCIATED
MALADIES.
SO, THE OTHER ASPECT OF THIS WAS
THAT WE WANTED TO CONTROL THIS
EXPERIMENT FOR A VARIETY OF
DIFFERENT TYPES OF DISEASE
STATES AS WELL.
SO, RATHER THAN JUST GET ACUTE
PANCREATITIS INDIVIDUALS AND
MATCH THEM WITH CONTROLS, WE
ACTUALLY WANTED TO MATCH THEM
WITH TWO ADDITIONAL DIFFERENT
TYPES OF CONTROLS.
SO, ONE OF THEM, WE WANTED TO
CONTROL AROUND FOR PANCREATIC
DISEASE IN GENERAL, RIGHT?
SO WE WANTED TO HAVE A CHRONIC
PANCREATITIS COHORT AS WELL
AS -- OR PART OF OUR COHORT, AS
WELL AS THE ACUTE PANCREATITIS
SECTION OF OUR COHORT.
SO, WE COULD CONTRAST THE TWO OF
THEM AND FIND OUT WHAT WAS
UNIQUE TO EACH ONE OF THOSE.
WE ALSO, YOU KNOW, THESE
INDIVIDUALS, THEY ARE IN PAIN,
RIGHT?
SO WE ALSO WANTED A PAIN CONTROL
AS WELL BECAUSE WE KNOW THERE
ARE OTHER MARKERS FOR PAIN THAT
CIRCULATE IN BLOOD AND IN URINE,
AND WE WANTED TO MAKE SURE THAT
WE WEREN'T LOOKING AT SOMETHING
THAT WAS JUST ASSOCIATED WITH
PAIN.
SO WE ALSO HAD A SECTION OF OUR
COHORT THAT WAS BROUGHT INTO THE
HOSPITAL FOR EXTREMITY FRACTURE,
SO BROKEN ARM OR BROKEN LEG, AND
SO FOR THAT WE HAD A COHORT THAT
WAS NOT EXCEPTIONALLY LARGE,
RIGHT?
OUR COHORT HERE IS AROUND 120,
130 INDIVIDUALS HERE.
130 INDIVIDUALS.
OF WHICH WE HAD 28 THAT WERE
ACUTE PANCREATITIS, AND 50
CHRONIC PANCREATITIS, THE REST
REMAINED FOR THE OTHER CONTROLS.
BUT WE WERE DOING SORT OF MORE
CLASSICAL BIOMARKER DISCOVERY
FOR THESE.
WE ALSO WANTED TO FOCUS ON
CONTROLLING FOR A BUNCH OF OTHER
TYPES OF VARIABLES, SO AS I
SAID, OBESITY IS ASSOCIATE D
WITH THE DISEASE, WE WANTED TO
CONTROL FOR BMI, MAKE SURE THEIR
AGE WAS ABOUT THE SAME AGE
RANGE, IF POSSIBLE.
THEIR HEIGHT AND WEIGHT WERE
ABOUT THE SAME HEIGHT AND WEIGHT
AS POSSIBLE.
WE WERE ABLE TO REALLY WELL
CONTROL I THINK FOR BMI, HEIGHT
AND WEIGHT.
BUT ONE OF THE THINGS THAT WAS
SORT OF IMMEDIATELY SURPRISING
TO US WAS ACTUALLY THE SORT OF
STRUCTURE OF THE COHORT ITSELF.
SO, TYPICALLY WHENEVER I'M GIVEN
AN EXPERIMENT WHERE THERE'S A
POTENTIAL COHORT FOR ME TO
STUDY, BEFORE I'M EVEN GOING TO
ACTUALLY TRY TO DIG INTO WHETHER
THIS IS -- WHETHER I'M GOING TO
MAKE MEASUREMENTS OR NOT TO
BEGIN WITH, I WANT TO HAVE SOME
UNDERSTANDING OF THE COHORT
ITSELF.
SO THE FIRST THING I DID WAS TRY
TO SEE WERE THERE DRIVERS OR
SEPARATIONS INSIDE THE COHORT
CONFOUNDING FACTORS THAT MIGHT
BE A PROBLEM.
ONE OF THE ONES THAT WAS
SURPRISING TO US, UP FRONT, WAS
EVEN WHEN WE TRIED TO CONTROL
FOR AGE AND FOR SEX, WHAT WE
FOUND WERE THAT THERE WAS A
LITTLE BIT OF A DEVIATION IN AGE
AND SEX AS A COMBINED VARIABLE.
THIS WAS SOMETHING THAT WAS SORT
OF SURPRISING TO US, BUT IT
SEEMED LIKE ACUTE PANCREATITIS
AND CHRONIC PANCREATITIS FEMALES
TENDED TO PRESENT SLIGHTLY LATER
THAN OUR MALE COHORTS, SO WE
WERE MATCHING SPECIFICALLY BASED
ON SORT OF THE EXPECTED AVERAGE
RANGE.
AND WE FOUND THAT THERE WAS
ACTUALLY SOME DEVIATION HERE.
SO THESE FEMALES ENDED UP BEING
A LITTLE BIT OLDER AS WELL.
SO THIS WAS SOMETHING WE HAD TO
CAREFULLY CONTROL FOR.
BUT I RECOMMEND WHENEVER YOU'RE
STARTING AN EXPERIMENT OF THESE
TYPES OF NATURES, FIGURE OUT
KIND OF ARE THERE OTHER
POTENTIAL CONFOUNDING FACTORS
BEFORE YOU EVEN BEGIN THE
EXPERIMENT, BECAUSE NOW THIS IS
SOMETHING WE CAN ACTUALLY START
CONTROLLING THE EXPERIMENT
AROUND.
SO, WHEN WE DESIGNED THE -- SO
WHEN WE DESIGNED THE EXPERIMENT
TO BEGIN WITH, ON THE MASS SPEC,
OUR SAMPLE ORDER ANALYSIS, FOR
EXAMPLE, THE FIRST THING THAT WE
DID WAS WE BLOCKED AROUND THIS
PARTICULAR PROBLEM, ORGANIZED IT
SUCH THAT WE DIDN'T HAVE LIKE
ALL OF THE CHRONIC FEMALES AT A
SPECIFIC SECTION IN OUR POOL.
SO WE WERE ABLE TO KIND OF FOCUS
IN ON SEPARATING THEM OUT BY AGE
AS WELL.
SO, THINKING ABOUT THESE THINGS
AS PARTICULARLY PROBLEMATIC, AND
WORTH TRYING TO INVESTIGATE.
SO, FROM A DIAGNOSIS
PERSPECTIVE, ACUTE PANCREATITIS
IS A LITTLE BIT CHALLENGING TO
DIAGNOSE.
WE WANTED TO MAKE SURE WE HAD A
REALLY COHERENT COHORT, AND SO
WE HAD A LONG LIFT WITH ALL OF
THE INDIVIDUALS, ALL OF THE
ACUTE PANCREATITIS INDIVIDUALS
WHO WERE DIAGNOSED WITH IMAGING
DATA AS WELL.
SO WE WERE ABLE TO SAY, OKAY,
WELL, THERE'S THESE INDIVIDUALS
DEFINITELY HAVE IT NOT JUST
BECAUSE OF ELEVATED LEVELS BUT
ALSO BECAUSE THEY HAD MORE FLUID
IN THE PANCREAS.
YOU CAN SEE THAT HERE.
THIS PANCREATIC BODY LOOKS MORE
LIKE THE LIVER BUT OVER HERE
IT'S A BIT DARKER IN THE ACUTE
PANCREATITIS INDIVIDUALS, THAT
MEANS MORE FLUID IN THE PANCREAS
AND IT'S SWOLLEN A LITTLE BIT.
WE USED THIS IMAGING AS
VALIDATION FOR TRYING TO MAKE
MEASUREMENTS.
SO, FOR THIS TYPE OF WORK, WE
WERE INTERESTED IN TRYING TO DO
ANALYSIS OF IT TO TRY TO
IDENTIFY POTENTIAL PROTEINS THAT
WOULD BE INTERESTING FOR MAKING
MEASUREMENT.
AND AS AN ANALYTICAL CHEMIST, I
WANTED TO APPROACH THIS PROBLEM
FROM A SLIGHTLY DIFFERENT
PERSPECTIVE THAN A NORMAL
MEASUREMENT PERSPECTIVE.
AND SO JUST TO KIND OF PUT
EVERYONE ON THE SAME FRAMEWORK,
I WANT TO TALK ABOUT WHAT A
NORMAL PROTEOMIC EXPERIMENT
TYPICALLY IS.
YOU GUYS HAVE ALL DONE
PROTEOMICS EXPERIMENTS BEFORE,
OR MASS SPECTROMETRY EXPERIMENT.
AND SO JUST TO KIND OF REFRESH
YOUR BRAINS, LIKE THE MOST
COMMON APPROACH TO TRYING TO DO
MASS SPECTROMETRY IS TO DO
DATA-DEPENDENT ACQUISITION.
WHEN YOU'RE THINKING ABOUT
ACQUISITION YOU'RE SEPARATING
PEPTIDES ACROSS RETENTION TIME.
EACH IS A DIFFERENT PEPTIDE
ELUDING OVER TIME AND YOU CAN
SEE THE RISE AND FALL OF THAT
PARTICULAR PEPTIDE IN THAT
DATASET.
SO, AS THE INSTRUMENT IS GOING
ALONG, IT'S CHUNKING THROUGH
TIME, COLLECTING THESE PRECURSOR
SCANS.
AND WAITS FOR ONE OF THESE PEAKS
TO RISE ABOVE A CERTAIN
THRESHOLD.
ONCE IT RISES ABOVE THE
THRESHOLD WE COLLECT A
SEQUENCING SPECTRUM FOR THAT
PARTICULAR PEPTIDE, AND WE STICK
IT ON AN EXCLUSION LIST.
WE'VE MEASURED THIS PEPTIDE,
DON'T NEED TO MEASURE IT AGAIN.
SO WE'LL PUT IT ON AN EXCLUSION
LIST FOR A CERTAIN PERIOD OF
TIME.
THEN THE INSTRUMENT WILL GO ON
THE NEXT ONE, PICK THE NEXT MOST
ABUNDANT SCAN, THAT'S THE NEXT
ONE I'M GOING SEQUENCING
SPECTRUM FOR, AND ANOTHER AND
ANOTHER, JUST TICKING THROUGH
TIME, RIGHT?
SO, THIS APPROACH IS EFFECTIVE
AS TRYING TO DIG DEEPLY INTO A
PARTICULAR EXPERIMENT, BECAUSE
IT USES EXCLUSION AS A WAY TO
SAY I DON'T NEED TO SAMPLE THIS
THING OVER AND OVER AGAIN, I
JUST WANT TO MEASURE ONCE AND BE
DONE WIT.
THE INSTRUMENT MAKES A LOT OF
DECISIONS AS PART OF THE
PROCESS.
OKAY, THIS IS MOST ABUNDANT PEAK
AT THIS SPECIFIC TIME AND
CONSEQUENTLY THERE'S SOME
STOCHASTICITY BUILD INTO THE
SYSTEM.
SO, MY LAB HAS BEEN FOCUSING ON
DATA INDEPENDENT ACQUISITION AS
A MODALITY FOR MAKING
MEASUREMENTS, SO WITH
DATA-INDEPENDENT ACQUISITION, WE
SELECT RANGES TO MAKE SEQUENCING
SCANS FOR.
WE'VE GOT A RANGE OF 500 TO 525,
ANOTHER 525 TO 550, TAKING THOSE
RANGES, INSTEAD OF SELECTING
INDIVIDUAL PEPTIDES FOR MAKING
MEASUREMENT, WE'RE
CO-FRAGMENTING THEM.
SO AT EACH TIME POINT WE'RE
MAKING ANOTHER SEQUENCING SCAN
OVER AND OVER AGAIN.
AND SO I COLOR-CODED THESE
PEPTIDE PEAKS ASSOCIATED WITH
THE PRECURSORS THAT THEY COME
FROM, BUT EACH ONE OF THESE IS A
FRAGMENT ION SIMILAR TO FRAGMENT
IONS HERE, JUST ACQUIRED OVER
TIME.
AND BECAUSE WE SEE THE RISE AND
FALL OF THAT PRECURSOR PEAK, WE
SHOULD SIMILARLY SEE A RISE AND
FALL OF THE FRAGMENT ION PEAKS
AS WELL, RIGHT?
AND SO WE ARE CO-FRAGMENTS
MULTIPLE PEPTIDES TOGETHER AT
THE SAME TIME WHICH MAKES FOR
FAIRLY COMPLICATED SPECTRA.
IF YOU INVESTIGATE ANY ONE OF
THESE SPECTRA THEY SEEM TO BE
EXTREMELY MESSY AND DIFFICULT TO
INTERPRET USING SORT OF MORE
STANDARD APPROACHES.
AND IT BECOMES KIND OF AN
INFORMATIC PROBLEM TO TRY TO
TEASE APART SORT OF WHAT PEAKS
ARE ASSOCIATED WITH ONE PEPTIDE
OVER ANOTHER PEPTIDE, RIGHT?
AND SO, FROM THIS PARTICULAR
CASE WE CAN SAY, OKAY, WELL,
THESE BLUE PEAKS ARE ASSOCIATED
WITH THIS PEPTIDE WHILE THE RED
PEAKS ARE ACTUALLY ASSOCIATED
WITH SOMETHING THAT'S COMPLETELY
DIFFERENT.
SOMETHING THAT'S NOISE ORIENTED.
AND SO THE WAY IN WHICH WE TEND
TO TALK ABOUT THIS DATA, LOOK AT
THIS DATA, SORT OF HEAD-ON.
WE LOOK AT THE RISE AND FALL OF
THOSE SPECIFIC PEAKS, AND WE
LOOK AT THEM AS CHROMATOGRAMS,
ACROSS RETENTION TIME RATHER
THAN ACROSS MASS SPACE SO THE
INTERPRETATION OF THIS DATA IS A
LITTLE BIT DIFFERENT THAN -- SO
WHEN I SHOW DATA IT WILL BE A
LITTLE BIT DIFFERENT THAN USUAL.
THE ONE BIG ADVANTAGE TO THIS
TYPE OF APPROACH IS THAT IT
ALLOWS US TO KIND OF MAKE
MEASUREMENTS OF PEPTIDES THAT
ARE CHALLENGING TO MAKE
MEASUREMENTS OF.
SO, FOR EXAMPLE, UP HERE WITH
DATA-DEPENDENT ACQUISITION THESE
TWO PEAKS NEVER ROSE ABOVE
THRESHOLD FOR MAKING A
MEASUREMENT.
AND THEY WOULD NEVER GET
SCANNED.
WHILE IN ANOTHER SAMPLE YOU
MIGHT SEE THEM RISE ABOVE THAT
THRESHOLD, COLLECT MEASUREMENT
FOR THEM.
HERE WE'RE GOING TO FRAGMENT
THOSE SPECIFIC PEAKS, NO MATTER
WHAT.
EVEN IF IT'S VERY LOW INTENSITY,
WE SHOULD STILL SEE SOMETHING OF
A SIGNATURE FOR IT.
AND THIS GIVES US A WAY OR SOME
VISIBILITY TO SEE THAT
SIGNATURE, EVEN IF THAT
SIGNATURE IS DEEP IN THE NOISE.
WE TEND TO THINK OF THIS TYPE OF
MEASUREMENT WHERE WE'RE
COLLECTING THIS GLOBAL DATA HERE
OF FRAGMENTATION ACROSS ALL TIME
AS KIND OF A BEAUTIFUL PICTURE,
RIGHT?
AS A MONA LISA HERE, RIGHT?
IN REALITY, LIKE AS A PICTURE OF
THE ENTIRE PORTRAIT OF THE
LANDSCAPE OF THAT SPECIFIC
IMAGE, MAPPING EVERY PARTICULAR
PIXEL FOR IT, IN REALITY THESE
TEND TO BE REALLY MUDDY, RIGHT?
THE MEASUREMENTS THEMSELVES
TENDING TO REALLY MUDDY BECAUSE
OF ALL OF THE OTHER PEPTIDES
THAT ARE PRESENT AT EXACTLY THE
SAME TIME.
FOR EXAMPLE, HERE WE HAVE TO
TELL THIS BLUE PEAK IS SEPARATE
FROM THIS PURPLE PEAK, SEPARATE
FROM THE PINK PEAK.
SEE ALL OF THESE DIFFERENT
PIXELS ARE BLENDED TOGETHER, WE
END UP WITH A REALLY BLURRY
IMAGE.
IT WOULD BE TRICKY TO TELL WHICH
FRAGMENT IONS ARE ASSOCIATED
WITH THAT PEPTIDE BUT ALSO WHICH
FRAGMENT IONS ARE QUANTITATED
FOR THAT PEPTIDE BECAUSE THEY
MAY BE ASSOCIATED WITH MULTIPLE
PEPTIDES AT THE SAME TIME.
AND SO, ONE OF THE APPROACHES TO
DEAL WITH THAT IS ONE THAT I'M
GOING TO TALK ABOUT THIS NEXT
BIT.
AND I WANT TO TALK ABOUT THIS
FROM THE STANDPOINT OF DOING AN
ANALYSIS USING WHAT WE CALL GAS
PHASE FRACTIONATION, ANALOGOUS
TO THE MEASUREMENT WE MADE
BEFORE.
SO THIS IS A PRECURSOR SCAN FROM
400 TO, SAY, 1,000.
AND THESE ARE THE DIFFERENT
FRAGMENT ION SCANS WE'LL COLLECT
ALONG THE WAY BEFORE WE HIT
ANOTHER CYCLE TO MAKE ANOTHER
MEASUREMENT.
OKAY?
SO THIS WOULD BE KIND OF LIKE
THE STRUCTURE OF OUR DIA
EXPERIMENT.
SO IN THIS PARTICULAR STRUCTURE,
WE'RE USING 24 M/Z WINDOWS, IN
OUR MEASUREMENTS USING SOMETHING
AKIN TO 16 M/Z WIDE WINDOWS,
STAGGERED TO OFF SET BY A
FRACTION.
ANOTHER MODALITY TO TRY TO MAKE
A MEASUREMENT OF THIS WOULD BE
TO TAKE THAT SAME SAMPLE AND
RATHER THAN ANALYZE THE FULL
PROFILE OF WHERE YOU FIND
PEPTIDES, ANALYZE JUST A
SUBSET.
RATHER THAN 400 TO 1,000,
MEASURE FROM 400 TO 500, LET'S
DO IT WITH 4 M/Z WIDE WINDOWS,
IF THEY ARE STAGRD, OFFSET BY
50%, IT'S POSSIBLE USING
MULTIPLEXING SOFTWARE TO
INTERPRET LATE AS IF WE WERE
DOING HALF OF THE WINDOW WIDTH.
SO 2 M/Z WINDOWS.
IF WE CAN GET DOWN TO TWO WE'VE
COLLECTED THE SAME QUALITY DATA
WITH DDA, SAME ISOLATION LEVEL
AS DDA, BUT WE'RE COLLECTING
OVER TIME SO YOU SEE EVERY
POSSIBLE PEAK AT THE APEX AS
WELL.
SO IT'S REALLY AKIN TO PRM DATA.
IT'S PRM DATA BUT WITH A GLOBAL
PRM.
PRM FOR EVERY POSSIBLE PEPTIDE
SCANNED IN THIS SORT OF WAY.
SO THAT OPENS UP SOME REALLY
INTERESTING WAYS OF TRYING TO DO
ANALYSIS.
THIS IS EXPENSIVE, RIGHT?
IF WE WANTED TO DO THIS ACROSS
THE ENTIRE MASS, WE NEED TO RUN
EVERY SIMPLE SIX TIMES.
REALISTICALLY THERE'S NO WAY MY
INSTRUMENT IS GOING TO WORK FOR
SIX TIMES -- WE ENDED UP DOING
150 MEASUREMENTS, NO WAY I CAN
DO SIX TIMES THAT AND HAVE THE
INSTRUMENT CONTINUE TO FUNCTION.
YEAH?
[
>> (INAUDIBLE).
>> WE DEVELOPED ENCYCLOPEDIA
SOFTWARE, ENCYCLOPEDIA IS A DIA
SEARCH TOOL ANALOGOUS TO
SOMETHING LIKE SPECTRUM, FOR
EXAMPLE, OPEN SOURCED THAT MY
LAB PRODUCES.
THIS TOOL IS DESIGNED AROUND
THIS TYPE OF EXPERIMENT
MODALITY.
NOW, BECAUSE IT'S OPEN SOURCE
AND FREELY AVAILABLE TO
EVERYONE, PEOPLE HAVE STARTED
WRAPPING IT UP INTO OTHER TYPES
OF SOFTAIRE.
SCAFFOLD DIA USES ENCYCLOPEDIA
UNDER THE COVERS.
SKYLINE HAS A MODULE THAT RUNS
ENCYCLOPEDIA IN THIS BACKGROUND
SO THAT IT CAN DO PEAK PICKING
AND ASSIGNMENT OUT OF DIA DATA.
WE LIKE TO MAKE THIS POSSIBLE IN
LOTS OF AVENUES.
SO IT'S A REALLY POWERFUL
TECHNIQUE AND SKYLINE HAS A
REALLY NICE WORKFLOW FOR THIS
SPECIFIC TYPE OF EXPERIMENT
WHERE YOU'VE GOVERNMENT SIX GAS
PHASE FRACTIONS.
AS I SAID, IT'S A REALLY
EXPENSIVE EXPERIMENT TO RUN.
BECAUSE YOU HAVE TO RUN SIX
INJECTIONS FOR EVERY TIME.
BUT THE QUALITY OF THE DATA IS
INCREDIBLE, RIGHT?
SO IF WE GO BACK TO OUR SORT OF
MOCKUP HERE, IF WE LOOK AT THE
GAS PHASE FRACTIONATION WE HAVE
NARROW WINDOWS, BECAUSE THEY ARE
2 MZ WIDE APART AFTER
DEMULTIPLEXING WE CAN GET EACH
PEPTIDE IN ISOLATION AT EACH
TIME POINT.
WE'RE DOING TARGETED EXTRACTION
FOR OUR PEPTIDES OF INTEREST,
ABLE TO IDENTIFY THEM IN
ISOLATION, WE KNOW EXACTLY WHAT
FRAGMENTATION PATTERN THEY HAVE
AND EXACTLY WHAT RETENTION TIME
THEY HAVE.
AND WE DO THIS FOR EVERY
POSSIBLE PEPTIDE SO WE CAN TAKE
WHICH EVER WINDOW WE'RE
INTERESTED IN, MAKE
IDENTIFICATIONS OUT OF IT AND
HAVE A LIST OF IDENTIFICATIONS.
SO, YEAH?
>> (INAUDIBLE)
>> POTENTIALLY YOU COULD.
I WOULD SAY I WOULDN'T DO THE
EXPERIMENT THAT WAY.
IF I WERE USING -- THE QUESTION
FOR THE PEOPLE ON ZOOM IS ABOUT
USING TMT ESSENTIALLY WITH DIA
IS IT A WAY TO GET TMT TO WORK
WITH DIA.
THEORETICALLY YOU COULD IMAGINE
THAT BUT YOU WOULD LOSE
ADVANTAGES OF TNT, LOSE ABILITY
TO COLLECT MS 3, SO MS 3
ISOLATION IS MORE POWERFUL THAN
MS 2.
WE HAVE RATIO COMPRESSION.
WE'RE NOT GETTING TO THE SAME
ISOLATION AS WITH MS 3s.
FOR MOST PEOPLE DOING TMT, DOING
MS 3s IS PROBABLY CRITICAL TO
THEM MAKING GOOD MEASUREMENTS TO
BEGIN WITH.
THERE ARE SOFTWARE THAT YOU CAN
USE TO DISENTANGLE IT BUT THEY
TEND NOT TO WORK AS WELL AS
ACTUALLY COLLECTING MS 3
MEASUREMENTS THEMSELVES.
THAT WOULD BE MY RECOMMENDATION
FOR IT.
I THINK THAT DIA WITH TMT IS
CHALLENGING AT BEST.
AND DANGEROUS AT WORST.
DESPITE THE LITERATURE THAT
EXISTS IN THIS AREA.
BECAUSE ALL PEPTIDES PRODUCE THE
SAME REPORTER ION REGION, SO
EVERY PEPTIDE PRODUCES THAT SAME
REPORTER ION REGION.
THEY ARE ALL GOING TO REPORT IN
THE SAME REGION AND THAT'S WHERE
YOU GET THAT RATIO COMPRESSION
BECAUSE AVERAGING THE SIGNALS
TOGETHER GETS YOU ESSENTIALLY
CLOSER TO THE AVERAGE, CLOSER TO
THE 1 TO 1 TO 1 RATIOS.
CRITICAL PART OF THIS IS THAT
EACH PEPTIDE THAT YOU SEE
ACTUALLY DOESN'T HAVE LIKE ONE
OR TWO PEPTIDES BEHIND IT.
THE PROTEOME GOES DOWN LIKE WE
THINK OF THE PROTEOME AS WHEN WE
MAKE A MEASUREMENT WE MIGHT MAKE
FOUR AS TO MAGNITUDE.
EIGHT IS MAGNITUDE IN CELL
LINES, BLOOD SAMPLES, IT GOES
DOWN FURTHER THAN EIGHT ORDERS
OF MAGNITUDE.
ALL THE GRAPHS, THAT DOESN'T
MAKE IT -- IT MAKES IT INTO THE
MASS MASS SPEC, YOU SEE VERY
LITTLE BLIPS, OTHER NOISE, OTHER
PEPTIDES PRODUCING THAT OTHER
SIGNAL.
IT JUST LOOKS LIKE NOISE BECAUSE
IT'S ALL DOWN AT THE BOTTOM OF
THE GRAPHS.
THEY ARE TINY PEPTIDES THAT
YOU'LL NEVER DETECT BUT THEY
HAVE A PRESENCE AND THEY ALSO
REPORT IN THE SAME REPORTER ION
REGION.
RIGHT?
CONSEQUENTLY, YOU NEED TO BE
ABLE TO DO SOME ADDITIONAL LEVEL
OF ISOLATION.
SO, DIA WITH TMT DOESN'T WORK
VERY WELL.
I WOULD SAY AT WORST CASE
DANGEROUS, BEST CASE
CHALLENGING.
OKAY?
BUT YOU DO GET ISOLATION, RIGHT?
YOU GET QUITE GOOD ISOLATION,
THAT GETS YOU TO THE LEVEL AS IF
YOU WERE DOING PRM MEASUREMENTS
USING GAS PHASE FRACTIONATION,
REALLY EXPENSIVE BUT REALLY
POWERFUL.
NOW WE'RE BASICALLY TAKING THAT
IMAGE AND CUTTING IT UP INTO
DIFFERENT PIECES AND MEASURING
EACH ONE OF THOSE DIFFERENT
PIECES WITH HIGH FIDELITY AND
STITCHING THEM BACK TOGETHER
WITH THESE SIX INJECTIONS BACK
TOGETHER, RIGHT?
SO BASICALLY WHAT WE'RE MAKING A
MEASUREMENT OF IS THIS ZOOMED-IN
VIEW OF THE PICTURE INSTEAD.
OKAY?
SO, OBVIOUSLY IT'S WAY TOO
EXPENSIVE TO DO ON EVERY SINGLE
EXPERIMENT.
BUT WE CAN LEVERAGE THIS IDEA TO
BUILD LIBRARIES.
SO WHEN WE'RE TRYING TO THINK
ABOUT BUILDING LIBRARIES, WHAT
WE ACTUALLY DO IS END UP
COLLECTING A BUNCH OF THESE
DIFFERENT MEASUREMENTS SO
DIFFERENT SAMPLES, WE'LL RUN
PROTEIN QUANT ON EACH ONE, DIA
MEASUREMENTS, WIDE WINDOW DIA
FOR EACH.
OF THOSE WE'LL TAKE A POOL OF
SAMPLES AND ANALYZE THIS USING
SIX GAS PHASE FRACTIONATION
EXPERIMENT.
FROM THIS PRM QUALITY DATA WE
DETERMINE EXACT LOCATION IN TIME
AND MASS FOR WHAT WE SHOULD BE
LOOKING AT, WE KNOW WHICH
FRAGMENT IONS ARE MOST ON OUR
INTENSE I, WHERE THEY ELUTE.
MORE POWERFUL THAN CHEMICAL
FRACTIONATION, TYPICALLY PEOPLE
WOULD DO HIGH PH REVERSE
FRACTIONATION FOLLOWED BY DDA TO
BUILD LIBRARIES.
YOU'LL DO 10, 30, 50, 100
FRACTIONS OF THAT, MULTIPLE
TIMES OVER AND OVER AGAIN.
BUT EACH ONE OF THOSE FRACTIONS
ACTUALLY CHANGES THE MATRIX THAT
LIVES BEHIND THAT PARTICULAR
PEPTIDE.
CONSEQUENTLY, THE RETENTION
TIMES SHIFTS NOT BY A LOT BUT BY
A MINUTE OR TWO IN A 90 OR
100-MINUTE GRADIENT.
BY 1 TO 2% YOU'LL GET RETENTION
TIME SHIFTS.
THE ADVANTAGE, WE'RE USING THE
INSTRUMENT AS FRACTIONATION
POOL, THE MAY -- MATRIX IS THE
SAME.
ELUTIONS ARE EXACTLY THE SAME AS
THEY WOULD BE IN THE WIDE WINDOW
DIA MEASUREMENTS.
SO, WE ACTUALLY KNOW THEIR
RETENTION TIME TO ABOUT 20
SECONDS, IN ACCURACY, ON OUR
MEASUREMENT.
SO WHEN WE'RE ACTUALLY MAKING
OUR MEASUREMENT, WE DETECT
PEPTIDES OUT OF HERE.
IN THIS PARTICULAR CASE WE WERE
ABLE TO DETECT OUT OF THE GAS
PHASE FRACTIONATION EXPERIMENT
OVER 2,000 PROTEINS, TYPICALLY
THIS IS WHAT PEOPLE THINK OF AS
ENTIRE COHORT, ENTIRE SIZE OF
THE URINE PROTEOME, RIGHT?
WITH SIX INJECTIONS HERE,
ESSENTIALLY FREE TO US BECAUSE
THE MASS SPECTROMETER IS DOING
THE WORK.
IT'S EASY TO MAKE THESE
MEASUREMENTS.
NOW WE KNOW COORDINATES FOR
THESE PEPTIDES.
WE HAVE TO LOOK THEM UP IN
SINGLE INJECTION EXPERIMENTS WE
KNOW WHEN IT IS ON OUR COLUMN
WHEN ELUTE, WHICH ARE THE
QUANTITATIVE IONS.
FROM THAT EXPERIMENT WE'RE ABLE
TO IDENTIFY 1700 PROTEINS
QUANTIFIED IN WIDE WINDOW DIA
MEASUREMENTS BECAUSE ALL WE'RE
DOING IS LOOKING THEM UP AND
APPLYING FALSE DISCOVERY
CORRECTION TO THE SIGNAL THAT WE
LOOK UP TO SEE WHETHER IT'S
BACKGROUND OR NOT.
WE KNOW EXACTLY WHERE TO LOOK
FOR THESE PARTICULAR PEPTIDES
THOUGH.
IN THIS PARTICULAR CASE, SO WHAT
CHEMICAL MODIFICATIONS WE SEARCH
FOR, IN THIS CASE WE ACTUALLY
DIDN'T SEARCH FOR ANY CHEMICAL
MODIFICATIONS.
HERE WHAT WE WERE DOING IN THIS
SEARCH WAS ACTUALLY JUST USING A
PREDICTED LIBRARY TO DO OUR
ANALYSIS, PREDICTED EVERY
POSSIBLE PEPTIDE IN THE HUMAN
PROTEOME, LOOKED FOR EVERY
PEPTIDE AND TRIED TO MAKE A
DETECTION THAT WAY.
WE DO THIS USING PEPTIDE-CENTRIC
SEARCH APPROACH.
AGAIN, THIS IS PART OF THE
ENCYCLOPEDIA WAY OF ANALYZING
DIA DATA.
YEAH?
>> (INAUDIBLE)
>> THAT'S AN INTERESTING IDEA.
SO I THINK THE WAY I'M GOING TO
ANSWER THAT IS WITH THE
DIFFERENT QUESTION THAN I
TYPICALLY GET ASKED ABOUT, WHY
DO THIS.
THE QUESTION IS, WHY DO WE POOL
IN THE FIRST PLACE, RIGHT?
WHAT DOES POOLING ACTUALLY GIVE
US AND SO DO WE END UP LOSING
PEPTIDES THAT WE WOULD OTHERWISE
NOT BE ABLE TO SEE IF WE HAD
ANALYZED THAT ONE SAMPLE
INDIVIDUALLY.
IS THAT CLOSE ENOUGH TO THE
QUESTION?
BECAUSE THAT WOULD BE LIKE WHY
NOT JUST ANALYZE THE AP COHORT
ON ITS OWN AND TREAT THAT ON ITS
OWN.
THE WAY ARATIONALIZE BY POOLING
HERE, WE HAVE DILUTED ALL OF AP
SAMPLES BY 1/4, ESSENTIALLY, SO
ANYBODY BIOMARKER THAT'S IN THE
AP SAMPLES NEEDS TO BE
DETECTABLE EVEN THOUGH IT'S BEEN
DILUTED BY A FOURTH IN ITS
SIGNAL.
BUT WHAT I WOULD SAY IS IF YOU
CAN'T IDENTIFY PEPTIDES HERE,
EVEN WITH 1/4 OF THE SIGNAL,
WHEN YOU'RE DOING PRM QUALITY
MEASUREMENTS YOU'RE NEVER GOING
TO SEE THEM IN WIDE WINDOW DIA
MEASUREMENTS.
AND IF YOU WERE ABLE TO SEE --
DETECT A PEPTIDE ONLY IN HERE
AND NOT IN HERE, I DON'T THINK I
WOULD TRUST THAT DETECTION.
I THINK IT'S MUCH MORE LIKELY TO
BE A FALSE POSITIVE THAN
ANYTHING ELSE.
SO THIS IS ONE OF THE REASONS I
THINK MAKING DETECTIONS DIRECTLY
FROM DIA CAN LEAD YOU ASTRAY, IF
YOU MAKE DETECTIONS DIRECTLY
FROM HERE I WOULD MUCH RATHER
MAKE DETECTIONS FROM HERE, FROM
THE GLOBAL -- FROM THIS GLOBAL
SIX-INJECTION PRM QUALITY DATA
BECAUSE I'M MORE CONFIDENT IN
THE DATA UP FRONT AND ISOLATION
OF THE DATA.
ANYTHING I CAN SEE ONLY IN THESE
SAMPLES WOULD MAKE ME NERVOUS,
RIGHT?
TO TRY TO SAY SOMETHING ABOUT.
AT LEAST FROM A GLOBAL
PERSPECTIVE.
>> (INAUDIBLE).
>> AND IT HAS TO DO -- SO
SPECTRONAUT DOESN'T SUPPORT
GLASS PHASE FRACTIONATION, THE
SOFTWARE IS DESIGNED TO SAY I
NEED TO HAVE THESE DIAGNOSIS
PEPTIDES IN MY SAMPLE TO DO
ALIGNMENTS, TO DO MY ASSESSMENT.
BECAUSE YOU NEED THOSE PEPTIDES,
THOSE PEPTIDES HAVE DIFFERENT
MASSES, SO YOU ONLY HAVE A
HANDFUL OF THOSE PEPTIDES IN
EACH ONE OF OUR FRACTIONS.
THAT'S THE PROBLEM WITH IT.
AND IT REQUIRES ESSENTIALLY
SOFTWARE IS DESIGNED TO REQUIRE
YOU TO BUY THEIR PEPTIDES, TO BE
ABLE TO RUN THE SOFTWARE.
IT'S BECAUSE THEY MAKE THE MONEY
OFF THE PEPTIDES, NOT THE
SOFTWARE, BUT THAT'S A TOTALLY
DIFFERENT STORY.
IT'S NOT A FUNDAMENTAL FACT OF
GAS PHASE FRACTIONATION OR DIA.
YOU DON'T NEED DIAGNOSIS
PEPTIDES TO DO DIA, IF YOU CAN
IDENTIFY THE MOST ABUNDANT
PEPTIDES, RIGHT?
JUST WITHOUT HAVING ANY
RETENTION TIME ASSESSMENT OF
THEM, YOU CAN USE THOSE
POTENTIALLY AS ANCHORS, FOR
EXAMPLE.
SO THAT'S HOW ENCYCLOPEDIA
WORKS.
IT ACTUALLY HAS A TWO-PASS
PROCESS, IT TRIES TO IDENTIFY
PEPTIDES NOT KNOWING ANYTHING
ABOUT RETENTION TIME FIRST AND
HAS TO MAKE DETECTIONS WITHOUT
RETENTION AND DISCOVERS THE
RETENTION TIME COORDINATES FOR
WHERE ALL THE PEPTIDES SHOULD
ACTUALLY LIVE, THROUGH THAT
FIRST PASS ANALYSIS, AND DOES A
SECOND PASS AFTERWARDS KNOWING
WHERE THOSE PEPTIDES SHOULD LIVE
AND MAKING A CALL AS TO WHICH
PEPTIDES ARE ACTUALLY PRESENT OR
NOT.
DOES THAT MAKE SENSE?
AS A RESULT, IT DOESN'T MATTER
WHICH PEPTIDES EXIST IN THAT
SAMPLE.
IT CAN MAKE ALIGNMENT.
EVEN IN GAS PHASE FRACTIONS YOU
CAN IDENTIFY TENS OF THOUSANDS
OF PEPTIDES IN EACH ONE OF THESE
FRACTIONS.
DOES THAT ANSWER YOUR QUESTION?
IT'S A SOFTWARE LIMITATION, NOT
A CONCEPTUAL LIMITATION.
>> [OFF MICROPHONE]
>> AGAIN, IT WOULD BE
CONCEPTUALLY POSSIBLE TO, BUT
BIOGONOSIS WANTS YOU TO BUY
THEIR PEPTIDES AND HAVE
LIMITATIONS FOR THAT PARTICULAR
REASON.
LET ME TELL WHAT YOU WE FOUND
FROM A PANCREATITIS PERSPECTIVE
AND ASK MORE QUESTIONS ABOUT DIA
AFTERWARDS.
THERE WERE A BUNCH OF ROBUST
BIOMARKERS.
WE HONED IN ON THESE SPECIFIC
SIX MARKERS, INTERESTED FROM A
GLOBAL PERSPECTIVE LOOKING AT
EYE -- ACUTE PANCREATITIS
VERSUS CONTROLS, THESE SEEMED
INTERESTING MARKERS INCLUDING
THE GOLD STANDARD, AMYLASE.
NOT ONLY SERUM AMYLASE, WE FOUND
A SPECIFIC ISOFORM OF AMYLASE,
PARALOG, NOT JUST AMYLASE IN
GENERAL.
WE'LL GET BACK TO THAT AGAIN.
>> [OFF MICROPHONE].
>> EXACTLY.
SO CRP IS MORE OF A SANITY
CHECK, CRP IS ELEVATED
INFLAMMATION, ASSOCIATED WITH
TONS OF DISEASES.
IT'S A TERRIBLE BIOMARKER FROM
THE STANDPOINT OF BIOMARKER
DISCOVERY.
WE KNEW THAT GOING IN, RIGHT?
AND SO WE WANTED TO KEEP IT IN
MIND TO SEE IT OUT, BUT IT'S NOT
GOING TO BE USEFUL AS A
BIOMARKER GOING FORWARD.
YOU'RE ABSOLUTELY RIGHT.
AT LEAST IN THIS COHORT IT WAS
DIFFERENT, ALTHOUGH IT WAS NOT
DIFFERENT ENOUGH.
SO THESE FIVE MARKERS WERE THE
ONLY ONES THAT WERE ACTUALLY
DIFFERENT ACROSS ALL OF THE
DIFFERENT COHORTS, IN DIRECT AB
COMPARISONS.
OF ALL THE PROTEINS WE DETECTED
RED ONES ARE POTENTIAL MARKERS,
ALL EXTREME VALUES.
WE COULD HAVE CONSIDERED ALL RED
ONES AS POTENTIAL MARKERS.
THE REASON WE HONED IN ON THESE
AND CRP IS AN OUTSIDER, AN AB
VERSUS EACH OF OUR DIFFERENT
CONTROL COHORTS, THESE WERE THE
ONLY ONES THAT WERE SIGNIFICANT
ACROSS ALL OF THOSE DIFFERENT
CONTROLS.
SO, KEEPING A TON OF DIFFERENT
TYPES OF CONTROLS IS USEFUL.
CP WASN'T PART OF THIS.
IT WAS UP IN THE EXTREMITY
FRACTURES AS WELL.
IT WASN'T AS YOU MIGHT IMAGINE
NOT A GOOD BIOMARKER.
OKAY.
SOME MARKERS ARE BETTER THAN
OTHERS.
CP WAS ONE OF THOSE.
OH, IT WAS UP IN CHRONIC
PANCREATITIS AS WELL.
SO, CP IS AN EXAMPLE OF LIKE NOT
A GREAT BIOMARKER.
IT IS ELEVATED.
BUT AMYLASE IS AN EXAMPLE OF
SORT OF WHAT WE HAVE BEEN KNOWN
TO SEE IN THIS TYPE OF COHORT
WHICH IS SLIGHTLY ELEVATED, BUT
NOT INCREDIBLY ELEVATED.
WHEREAS OUR MARKER THAT WE FOUND
HERE AS OUR PRIMARY MARKER OF
INTEREST, CELA 2A WAS MORE
ELEVATED, HERE WE HAVE A 60-FOLD
ELEVATION IN URINE, THIS IS A
MARKER FOR GENERAL INFLAMMATION.
THESE ARE SUCCESSFUL TO A
DEGREE.
MORE INTERESTING FROM BIOLOGY
PERSPECTIVE AN BIOMARKER
DISCOVERY PERSPECTIVE, THIS ONE
MARKER IS THE ONE WE'RE
INTERESTED IN FROM A BIOMARKER
PERSPECTIVE.
THESE THREE OTHER ONES ARE ALSO
ASSOCIATED WITH PANCREATIC
CANCER.
THE SUPER INTERESTING, RIGHT?
PROGRESSION IN DISEASE, THERE'S
A HYPOTHESIZED PROGRESSION IN
DISEASE FROM ACUTE PANCREATITIS
TO CHRONIC PANCREATITIS TO
PANCREATIC CANCER AND SEE AS
PRIMARY MARKERS OF THE OTHER
THREE THESE ARE ASSOCIATED WITH
THAT PROGRESSION RATHER THAN
MAYBE THE DISEASE ITSELF.
WHICH IS INTERESTING -- YEAH?
>> [OFF MICROPHONE]
>> NO, NO, NO, WE SEE THESE AS
MARKERS AS INCREASING IN CHRONIC
PANCREATITIS SIGNATURES.
SORRY, THERE'S SIGNATURES, WHEN
THEY ARE INCREASED THEY
TYPICALLY ARE GOOD MARKERS FOR
PANCREATIC CANCER.
OKAY.
AND THAT FOLLOWS OUR DATA AS
WELL.
SO, ELASTASE TRACKS WELL WITH
AMYLASE, THIS IS ELASTASE HERE,
AMYLASE HERE.
ONE THING THAT'S CLEAR, THE
SEPARATION, RIGHT, BETWEEN THE
AP COHORT AND NON-AP COHORT IS
QUITE WIDE WHEN WE'RE LOOKING AT
ELASTASE AND NARROW WHEN LOOKING
AT AMYLASE AS A MARKER IN
PARTICULAR.
SIMILARLY IF WE'RE THINKING FROM
A BIOMARKER DIAGNOSTIC
PERSPECTIVE, AMYLASE IS LESS
SUCCESSFUL THAN ACTUALLY USING
AMYLASE IN COMBINATION WITH OUR
MARKER OF CHOICE.
WHERE OUR MARKER IS THE ONE THAT
SORT OF DRIVES THE SEPARATION IN
PARTICULAR.
JUST TO KIND OF GO THROUGH SOME
OF THE THIS QUICKLY, WE TESTED
ANOTHER 30 INDIVIDUALS AT A
DIFFERENT TIME, ANOTHER SET OF
SAMPLES WHERE WE WERE ABLE TO
REPRODUCE THIS MEASUREMENT AS
WELL IN SORT OF MORE TARGETED
INTERPRETATION OF DIA DATA.
SO, IN AP CP HEALTHY COHORT
WITHOUT EXTREMITY FRACTURE, A
SEPARATE BATCH, CELA2A IS ONE OF
THE HIGH MARKERS FOR THIS
DISEASE.
AMYLASE IS NICELY SEPARATED.
WE SEE MULTIPLE FORMS OF AMYLASE
TOO.
AND SOME OF THEM HAVE DIFFERENT
LEVELS OF MARKER ACTIVITY THAN
OTHERS, RIGHT?
WHEN YOU MAKE AN ELISA AND USE
AN ANTIBODY FOR A READOUT, IT'S
MEASURING ALL OF THEM, RIGHT?
IT'S MEASURING THEM BASED ON
SHAPE, NOT BASED ON SEQUENCE OR
IDENTITY TO A SPECIFIC ISOFORM.
WHEN YOU'RE MEASURING THEM THIS
IS THE MARKER THAT IS PROBABLY
THE MOST PRECISE, AMY2A, CRP IS
AGAIN ELEVATED COME WHAT.
WE ALSO VALIDATED THIS IN A
DIFFERENT WAY TOO WITH BASELINE
SO INDIVIDUALS COME IN WITH
ACUTE PANCREATITIS, WE MAKE A
MEASUREMENT, THREE MONTHS LATER
DO A FOLLOW-UP MEASUREMENT,
RIGHT?
SO HERE ONE OF THE THINGS WE
WOULD LIKE TO SEE, HERE ARE SOME
SIGNALS DROP, WE SEE SOME OF
THEM DROP BUT NOT ALL OF THEM
DROP.
THAT'S INTERESTING TO US AS
WELL.
SO ALSO TRUE FOR AMYLOIDS AND
CRP.
WE THINK THAT SOME OF THESE
INDIVIDUALS ACTUALLY ARE, YOU
KNOW, IN ACUTE PANCREATITIS THEY
WILL GET AN ATTACK AND THEN
FREQUENTLY WILL GET A RECURRENT
ATTACK AGAIN LATER.
THAT RECURRENT ATTACK WILL BUILD
INTO CHRONIC PANCREATITIS
AFTERWARDS THAT.
THAT PATHWAY ARE KNOWN, WE'RE
CONCERNED THESE ARE SLATED TO
GET CHRONIC PANCREATITIS IN THE
FUTURE.
THIS IS FOLLOW-UP WORK FOR
LATER.
JUST TO GIVE YOU SOME FEELING
FOR THIS, IF WE LOOK AT OUR
MARKERS ELASTASE VERSUS AMYLASE,
THIS IS CHANGED.
A FOLD CHANGE FROM BASELINE TO
FOLLOW-UP MEASUREMENT.
IN GENERAL FOLLOWING ON KIND OF
A LINE WITH SOME OUTLIERS HERE,
WHICH STRUCK US AS UNUSUAL,
SOMETHING HARD TO EXPLAIN HOW
CAN SOMEONE HAVE THEIR HIGH
ELASTASE WITHOUT HAVING HIGH
AMYLASE AS WELL?
WE'RE NOT TOTALLY SURE ABOUT THE
DETAILS OF THAT.
WE DO KNOW THAT ELASTASE FOLLOWS
QUITE CLEANLY WITH CRP LEVELS SO
MIGHT BE THESE PEOPLE HAVE SOME
SORT OF VESTIGIAL OR LATENT
PANCREATIC DISEASE STILL, BUT
THEY ARE NOT EXPRESSING IN THE
SAME SORT OF WAY AS WITH
AMYLASE.
OKAY.
SO, I SHOWED YOU THIS LIST OF
BIOMARKERS AT THE BEGINNING.
AND ONE OF THE BIOMARKERS THAT I
HAD LISTED HERE IS ACTUALLY
CALLED PANCREATIC ELASTASE
ALREADY.
AND SO WHEN I SAW THIS, IN THE
LITERATURE, OH, MAN, WE JUST
FOUND SOMETHING THAT SOMEONE
ELSE FOUND A WHILE AGO.
AND PANCREATIC ELASTASE IS CELL
1A, DIFFERENT SEQUENCE,
DIFFERENT PROTEIN, DIFFERENT
CHAIN.
DESPITE ITS NAME, PANCREATIC
ELASTASE, IT ORIGINATES SO WE
SEE IT IN STOOL, WE DON'T SEE IT
IN URINE OR IN BLOOD.
IT'S ONLY ASSOCIATED WITH STOOL.
AND SO WHILE IT'S BEEN CALLED
PANCREATIC ELASTASE, IT'S
ACTUALLY NOTED IN UNIPROTE
DOESN'T ORIGINATE FROM THE
PANCREAS.
THIS SHOWS WORSE LEVELS OF
ACTIVITY IN THESE OLDER PAPERS
SO THIS IS FROM 1998.
SO, AT THE TIME THEY WERE LIKE,
OKAY, THIS IS AN INTERESTING
MARKER, BUT IT DOESN'T WORK AS
WELL AS AMYLASE, WE'RE GOING TO
THROW IT OUT.
AND SO THEY STOPPED WORKING ON
THIS AREA.
AND SO ONE OF THE THINGS THAT WE
THINK IS INTERESTING, SO IN OUR
DATASETS IN URINE AND IN PLASMA
WE ACTUALLY DON'T SEE THIS
PROTEIN AT ALL, RIGHT?
SHOULD BE NOT FOUND, ONLY FOUND
IN STOOL.
BUT IT IS PRESENT IN THESE
EXISTING DATASETS BECAUSE AGAIN
THEY ARE USING ANTIBODIES,
RIGHT, FOR MEASUREMENT,
ELISAs.
IF YOU LOOK AT SEQUENCE
HOMOLOGY, CELL 1A AND 2A, THEY
ARE SIMILAR ENOUGH AT THE
SEQUENCE LEVEL, BUT DIFFERENT
ENOUGH THAT WE CAN DEFINITELY
TELL THE DIFFERENCE, RIGHT?
WITH MASS SPEC, DIFFERENT
PROTEINS, WE'RE NOT GOING TO
CONFUSE THE TOO.
BUT ANTIBODIES ARE NOT BINDING
TO SEQUENCE BUT TO THE SHAPE OF
THE PROTEIN.
THESE WHO PROTEINS HAVE
ESSENTIALLY THE SAME PHYSICAL
SHAPE.
AND BECAUSE OF THAT, THE
ANTIBODIES ARE PROBABLY
CROSS-REACTIVE.
WE THINK WHAT THIS PERSON WAS
FINDING AS URINE BIOMARKER IN A
PROTEIN SUPPOSED TO ONLY EXIST
IN STOOL WAS ACTUALLY THE
PROTEIN THAT WE FOUND.
IF THEY HAD DONE FOLLOW-UP OR
BETTER TYPE OF ASSAY, THEY WOULD
HAVE ACTUALLY FOUND THE RIGHT
PROTEIN AS THE MARKER, AND WOULD
HAVE ACTUALLY HAD A MUCH BETTER
ASSAY 30 YEARS WHEN THIS STUDY
WAS FIRST DONE.
AGAIN, THESE ANTIBODIES BECOME
REALLY PROBLEMATIC WHEN WE USE
THEM AND REQUIRE THEM AS
READOUTS FOR A LOT OF THESE
BIOLOGICAL MEASUREMENTS.
WE HAVE TO BE ABLE TO BUILD
ASSAY TOOLS THAT ARE MORE ROBUST
THAN SINGLE ANTIBODY BINDING,
RIGHT?
OKAY.
SO I WANTED TO TOUCH BRIEFLY ON
THIS NOTE AGAIN.
ACUTE PANCREATITIS BECOMES
RECURRENT PANCREATITIS, BECOMES
CHRONIC PANCREATITIS, BECOMES
CHRONIC PANCREATIC SIGNATURE, A
HYPOTHESIZED IDEA.
WE ARE DIGGING INTO THIS A
LITTLE BIT MORE BECAUSE WE HAD A
CP COHORT WITH AP COHORT, CP
PROTEINS ARE ALREADY INTERESTING
FROM THAT PERSPECTIVE.
TOP SIX CP PROTEINS SPECIFIC TO
CP ALONE, OF THESE SIX FIVE HAVE
ALREADY BEEN ASSOCIATED WITH
PANCREATIC CANCER AS WELL.
SO, FROM THIS PERSPECTIVE, WE
SEE THIS COINCIDENCE IS WORTH
DIGGING INTO MORE SERIOUSLY, AS
A TRUE PROGRESSION THAT COMES
OUT OF THE DATASET.
CHRONIC PANCREATITIS IS EASIER
TO DIAGNOSE, SO THIS IS TOP
MARKER OF THIS.
FROM ITS IDENTIFICATION RATE,
IT'S ALMOST A PERFECT BIOMARKER
FOR THIS TYPE OF MEASUREMENT.
WE'RE FOLLOWING UP ON THIS AS A
SEPARATE STUDY THAT WE'RE GOING
TO BE WORKING ON IN THE FUTURE.
BUT THAT ONE IS STILL LEFT TO BE
DISCUSSED.
THAT SECTION.
BUT JUST IN THE LAST FEW MINUTES
I WANT TO OUTLINE A COUPLE
DIFFERENT CONCLUSIONS HERE.
SO ONE OF THEM IS THAT URINE IS
A REALLY POWERFUL SOURCE FOR
THINKING ABOUT BIOMARKERS.
WE DON'T TYPICALLY THINK ABOUT
BIOMARKERS IN URINE BECAUSE
ASSAYS TEND NOT TO WORK WELL FOR
THEM BECAUSE OF VARIATION IN
LEVEL IN URINE.
IF YOU HAVE A THOUSAND-FOLD
VARIATION IN THE PROTEIN
ABUNDANCE IN URINE, TRYING TO
SEE YOUR PROTEIN OF INTEREST IN
THAT NOISE IS REALLY
CHALLENGING.
THE FIRST THING WE DO IN TRYING
TO DO A PROTEOMIC ASSESSMENT OF
IT IS TO DO -- TO TRY TO CRASH
OUT THE PROTEIN TO BEGIN WITH.
BECAUSE OF THAT WE'RE ALREADY
NORMALIZING BASICALLY ON THE
PROTEIN ABUNDANCE AS THE FIRST
STEP IN OUR PRACTICE FOR THIS.
SO, WHILE IT DOESN'T WORK REALLY
WELL FOR TRADITIONAL ASSAYS,
USING ELISAS OF DIRECT URINE WE
THINK IT WOULD WORK WELL
ELISAs OF PROTEIN.
AP WE'RE FOLLOWING UP ON FIRST,
CP HAS A STORY WE'RE COMING OUT
WITH LATER THAT WE'RE WORKING ON
RIGHT NOW.
THE LAST THING IS THAT WE DO SEE
THIS STRONG PROGRESSION FROM AP
TO CP TO PANCREATIC CANCER,
DRIVERS FOR CANCER, AND SO THIS
I THINK IS A DIFFERENT AVENUE
FOR US TO START INVESTIGATING
THE EFFECTS OF THESE SPECIFIC
TYPES OF PROTEINS FROM A
MECHANISTIC PERSPECTIVE, NOT
JUST BIOLOGICAL PERSPECTIVE.
OKAY.
WITH THAT I WANT TO ACKNOWLEDGE
MY COLLABORATORS, THIS WORK
WOULD NOT BE POSSIBLE WITHOUT
THE COLLABORATION FROM
CINCINNATI CHILDREN'S.
SO MAISAM HAS BEEN CRITICAL IN
TRYING TO DO THIS.
WE'VE BEEN ACTING AS PARTNERS
THROUGHOUT THE ENTIRE PROCESS.
TWO MEMBERS OF MY LAB ARE
INVOLVED IN THIS WORK.
MADDIE AND ARIANA, COLLABORATORS
AT OSU, VENKATA IS THE FIRST
AUTHOR, DRIVING INTERPRETATION
OF DATA AFTERWARDS.
SO, WITH THAT I THINK WE HAD A
LOT OF QUESTIONS DURING.
I'M HAPPY TO TAKE OTHER
QUESTIONS TOO.
BUT WE CAN CLOSE.
>> [OFF MICROPHONE].
>> YEAH, I DON'T ANY.
>> [OFF MICROPHONE]
>> SO, WHAT DO WE DO ABOUT BATCH
EFFECTS? THE FIRST THING IS
MAKE SURE WE DON'T HAVE BATCH
EFFECTS TO BEGIN WITH.
AND SO, THE FIRST THING I SHOWED
YOU AT THE BEGINNING WAS THOSE
BOX PLOTS, RIGHT?
THE BOX PLOTS ARE A SIMPLE
BIOINFORMATIC APPROACH TO SAY IS
THERE ANY CONFOUNDING VARIABLE I
HAVEN'T THOUGHT ABOUT YET, IS
THERE ANY CORRELATION OF ONE
VARIABLE WITH ANOTHER VARIABLE
THAT I HAVEN'T THOUGHT ABOUT
YET.
AND I THINK IT'S ALWAYS
WORTHWHILE BEFORE YOU BEGIN AN
EXPERIMENT TO TRY TO TEASE APART
WHAT MIGHT BE A CONFOUNDING
FACTOR BEFORE YOU BEGIN, AND
MAKE SURE THAT BECOMES PART OF
HOW YOU DO BATCHING TO BEGIN
WITH.
SO YOU DON'T WANT ALL OF ONE
TIME OF -- TYPE OF SAMPLE IN ONE
TYPE OF BATCH.
IT MAY BE CRYPTIC, NOT OBVIOUS
THIS IS THE SAMPLE THAT'S A
PROBLEMATIC SAMPLE.
IT WASN'T JUST THE CP
INDIVIDUALS.
IT WAS SPECIFICALLY THE CP
INDIVIDUALS THAT WERE FEMALE
THAT WERE OLDER, RIGHT?
THOSE WERE A DIFFERENT COHORT
ALMOST, DIFFERENT GROUP IN OUR
COHORT THAT WE NEEDED TO MAKE
SURE THEY WERE SPREAD ACROSS THE
EXPERIMENT.
STEP 1, TRY TO AVOID THE
POTENTIAL PROBLEM.
STEP 2, THAT WE DO, WE ALWAYS
TRY TO MONITOR HOW THE
INSTRUMENT IS PERFORMING OVER
TIME.
SO THAT WE CAN MAKE SURE THAT WE
ARE SUCCESSFULLY MAKING THE SAME
NOT JUST DETECTIONS BUT THE SAME
INTENSITY MEASUREMENTS.
ALL OF THE MEASUREMENTS WE'RE
MAKING HERE ARE BASED OFF
INTENSITY NOT BASED ON WHETHER
WE DETECTED THAT PARTICULAR
PEPTIDE OR NOT, AS WOULD YOU
WITH DDA.
OUR EQUAL CONTROL ISN'T RUNNING
STANDARD HeLa.
YOU CAN STILL DETECT THE SAME
AMOUNT OF PROTEINS FROM A
HeLa DIGEST.
WE SEE DEGRADATION IN INTENSITY
FAR EARLIER, USUALLY THAT
HAPPENS WITHIN A COUPLE WEEKS.
YOU START TO SEE DIRTYING OF
CERTAIN PARTS OF THE INSTRUMENT,
USUALLY INPUT CAPILLARY IS THE
FIRST THING THAT GETS DIRTY.
AND THAT ACTUALLY TYPICALLY
DOESN'T AFFECT YOUR DETECTION
RATE.
IT JUST AFFECTS INTENSITIES OF
YOUR FRAGMENT IONS.
THAT'S ONE ADDITIONAL ASPECT.
WE TRY TO MAKE SURE SAMPLES
AFTER THEY HAVE BEEN BATCHED
TOGETHER CAN GET ACKNOWLEDGIZED
QUANTITATIVELY AS A GROUP.
I DIDN'T SHOW YOU A SLIDE
TALKING ABOUT BATCHING BUT WE
TRY TO MAKE SURE ALL SAMPLES CAN
BE NORMALIZED TOGETHER.
IF THEY CAN'T FROM A GLOBAL
PERSPECTIVE WE WILL EITHER THROW
OUT A SAMPLE OR MARK IT AS BEING
PROBLEMATIC.
SO, THE WAY WE DO THAT IS
ACTUALLY LOOK AT, AGAIN, BOX
PLOTS OF THE GLOBAL INTENSITY
MEASUREMENTS THAT WE'RE MAKING
FOR ALL OUR PROTEINS.
IF THEY ARE STAYING CONSISTENT
WE CAN SAY THESE SAMPLES DON'T
NEED TO BE SORT OF ADDITIONALLY
NORMALIZED TOGETHER BEYOND THE
LEVEL OF NORMALIZATION WE'VE
ALREADY EMPLOYED.
BUT THERE ARE WAYS OF CORRECTING
ONE BATCH VERSUS ANOTHER BATCH,
IF YOU NEED TO.
YOU JUST TYPICALLY IF YOU CAN
GET AWAY WITH NOT DOING IT,
HAVING FEWER CORRECTIONS IS
USUALLY SAFER.
DOES THAT ANSWER YOUR QUESTIONS?
COOL.
>> [OFF MICROPHONE]
COULD YOU DISCUSS WHAT WE CAN DO
TO CONTROL FALSE DISCOVERY RATE
WITH DIA, WHAT PUBLICATION
PROTOCOL SOFTWARE METHODS?
>> LET'S -- WHAT I WOULD SAY,
I'LL EXPLAIN THE PROBLEM AND
THEN WE CAN TALK ABOUT KIND OF
DIFFERENT APPROACHES OFFLINE.
THAT MIGHT BE THE BEST SOLUTION.
BUT THE PROBLEM I THINK IS
REALLY INTERESTING.
WE USE TARGET DECOY AS AN
APPROACH FOR DOING DIA ANALYSIS,
HOW HE WE DO WITH DDA.
YOU SEARCH AS MANY TARGETS AS DO
YOU DECOYS, YOU SAY, I WANT MY
TARGETS TO SCORE BETTER THAN
DECOYS.
A GREAT WAY TO SAY DECOYS
REPRESENT WRONG MATCHES AND I
WANT SUFFICIENT NUMBER -- I WANT
AS FEW DECOYS TO KIND OF BE
ACCEPTED AS PART OF MY BATCH.
SO, DECOYS AS I SAID REPRESENT
WRONG MATCHES, RIGHT?
VERY MUCH WRONG MATCHES.
REVERSE SEQUENCES, WON'T
FRAGMENT AT ALL ALIKE.
SO THE PROBLEM WITH TARGET DECOY
IS IT CONTROLS FOR WRONG
MATCHES.
NOT FOR KIND OF WRONG MATCHES.
AND THERE'S A CLASS OF KIND OF
WRONG MATCHES THAT BECOME A
PROBLEM WITH DIA.
SO, WITH DIA, YOU END UP HAVING
WIDER PRECURSOR ISOLATION
WINDOWS WHICH ALLOW PEPTIDES TO
FALL IN THAT WINDOW.
FOR YOUR DIA WORK WHAT'S THE
WINDOW YOU TYPICALLY USE?
SORRY?
7.5 WINDOWS?
WHAT'S YOUR CYCLE TIME IN THAT?
SO YOUR POINTS ACROSS THE PEAK
ARE NOT VERY GOOD.
YOU'VE GOT LIKE 60-SECOND LENGTH
PEAKS?
YEAH, NO, THE PEAK WIDTH.
HOW MANY POINTS ACROSS THE PEAK
DO YOU GET?
WE COULD TALK ABOUT THAT
AFTERWARDS.
7.5, RIGHT?
WHAT FITS IN 7.5?
WELL, OXIDATION, A SHIFT OF
METHYL WOULD FIT IN 7.5.
HOW MANY SEQUENCE VARIANTS ARE A
SHIFT OF METHYL?
MOST OF THEM.
MOST ARE THEM TO VALENE, THOSE
FALL IN THE ISOLATION WINDOW AND
BECOME POTENTIALLY A PROBLEM
BECAUSE NOW YOU HAVE A DIFFERENT
SOURCE OF FALSE DETECTIONS, THEY
ARE FALSE DETECTIONS THAT ARE
ALMOST THE SAME PEPTIDE BUT NOT
THE SAME PEPTIDE.
SO IT'S VERY EASY FOR US TO
OVERCALL IN OUR DETECTIONS.
WE SEE THIS ALL THE TIME WITH
SEQUENCE VARIANTS, EVEN WHEN YOU
REQUIRE A PRECURSOR SIGNAL YOU
STILL END UP IN CIRCUMSTANCES
WHERE YOU CAN MAKE ERRORS
ASSOCIATED WITH HAVING MOSTLY
THE RIGHT SEQUENCE BUT NOT
EXACTLY THE RIGHT SEQUENCE
BECAUSE OF THE WIDER PRECURSOR
ISOLATION WINDOW.
NOW, ONE WAY TO SORT OF DEAL
WITH THAT IS BY USING THE GAS
PHASE FRACTIONATION APPROACH,
TWO M-Z WIDE WINDOWS, NOW YOU
CAN SAY, WELL, I COULD MISS
ISOLUCING, MISSING DEAMIDATION,
WHATEVER THE MASS SHIFT IS HAS
TO BE REALLY SMALL.
WE WANT TO GET AT LEAST WITHIN
FOUR M/Z WIDE WINDOWS BEFORE WE
START ESCAPING THESE TYPES OF
PROBLEMS.
THIS IS A SOURCE OF FALSE
DISCOVERIES THAT LOOKS DIFFERENT
THAN WRONG PEPTIDES.
THESE ARE RIGHT PEPTIDES BUT
THEY ARE NOT EXACTLY THE RIGHT
PEPTIDE.
LET ME GIVE AN IDEA FOR HOW BAD
OF A PROBLEM THIS IS.
SO IN 12 M/Z WIDE WINDOWS SIZES
EVERY PEPTIDES THAT 20% CHANCE
OF SEQUENCE SIMILAR PEPTIDE IN
THE SAME WINDOW.
0% -- 20%.
THEY ALL HAVE THE SAME CHEMICAL
STRUCTURE ESSENTIALLY, BECAUSE
THEY ALL ARE ROUGHLY THE SAME
AMINO ACID, THEY ALL ELUTE AT
THE SAME TIME PERIOD AS WELL.
SO BECAUSE OF THOSE CONFOUNDING
FACTORS WE END UP WITH HAVING
CHALLENGES MAKING CALLS AS TO
WHICH ONE IS ACTUALLY PRESENT
AND MOST SOFTWARES WILL CALL
BOTH AS BEING PRESENT.
THAT'S WHERE WE GET SOURCE OF
OVERREPORTING AND OVERDETECTION.
DOES THAT ANSWER?
OKAY.
>> [OFF MICROPHONE].
>> I THINK THAT MS1s, USAGE OF
MS1s IN THIS ARE BENEFICIAL
BUT DON'T SOLVE THE PROBLEM.
WE SEE LOTS OF CIRCUMSTANCES
WHERE YOU HAVE AMINO ACID FLIPS,
INSIDE THE HUMAN GENOME.
THE HUMAN GENOME IS SO MASSIVELY
OVERREPEATED, RIGHT?
EVOLUTION NOT SOME SORT OF
HIGHERING BEING THAT KNOWS AND
PLANS, RIGHT?
IT COPIES GENES, EDITING THEM
SLIGHTLY, CALLING IT A NEW
PROTEIN, RIGHT?
BECAUSE OF THAT WE GET SO MANY
SUBTLE CHANGES IN OUR
CIRCUMSTANCES.
WE OFTEN SEE LIKE REVERSED
SEQUENCES OR SEQUENCE VARIANT
OVER HERE REPLACED BY THE
DIFFERENT SEQUENCE VARIANT OVER
THERE WHERE THEY HAVE THE SAME
PRECURSOR MASS.
ALSO PRECURSOR SPACE IS FLOODED
WITH OTHER TYPES OF PEPTIDES,
ESPECIALLY WHEN HAVE YOU VERY
SMALL GRADIENTS.
>> [OFF MICROPHONE].
>> HOW DO YOU CONTROL CARRYOVER,
YOU MEAN FROM ONE INJECTION TO
THE NEXT?
THAT'S A CHALLENGE.
I THINK PART OF THIS HAS TO DO
WITH LC ITSELF.
CARRYOVER IS CAUSED BY LC.
AND BY SORT OF WHATEVER IS
REMAINING EVEN AFTER A WASH ON
THE LC.
YOU GET CARRYOVER ON THE COLUMN
AS WELL SO PEPTIDES THAT COME
OFF LATER IN THE COLUMN, THE
SECOND TIME YOU RUN A GRADIENT
THROUGH I BOTH THINGS HAPPEN,
CARRYOVER AROUND 1% WE ESTIMATE.
SO 1/100 OF OUR MEASUREMENT.
SO ANY IDENTIFICATION THAT WE
CALL THAT IS 1/100 OF A
MEASUREMENT AWAY FROM WHERE IT
IS ON AVERAGE, WE ESSENTIALLY
SAY THAT'S CLOSE ENOUGH TO ZERO
TO BE ZERO.
FROM THAT PERSPECTIVE, WE'LL
CALCULATE A RATIO, WE KNOW THAT
BEYOND ESSENTIALLY 1/100 WE HAVE
RATIO COMPRESSION.
IT'S BETTER THAN EYE TRACK 1 TO
2 OR 1 TO 3 CHANGES.
HERE AS LONG AS WE'RE WITHIN 1
TO 100, ANYTHING OUTSIDE WE'RE
LIKELY TO SEE PROBLEMS WITH.
IN REPEATED INJECTIONS.
YOU CAN RUN BLANKS IN BETWEEN TO
CLEAN OUT COLUMNS BUT IN MANY
CASES WHERE THERE HAVE BEEN
MARKERS WE CARE ABOUT, THERE ARE
MANY TYPES OF PEPTIDES THAT
STICK TO COLUMNS, AND IN ONE
EXPERIMENT I RAN SIX OR SEVEN
BLINKS AND WAS STILL ABLE TO
DETECT COMING OFF THE COLUMN,
ELUTING OFF THE COLUMN.
AT THE SAME TIME WE WOULD STILL
HAVE TO DETECTIVE ARE THESE
ISSUES WITH DDA OR ANY OTHER
TYPE OF PROTEOMIC MEASUREMENT.
>> [OFF MICROPHONE].
>> AH, YES.
SO, THAT'S A GREAT QUESTION.
HOW DO WE DO PROTEIN EXTRACTION?
IT'S A CHALLENGE.
WE FEEL LIKE THAT'S OUR SECRET
CAUSE RIGHT NOW IS BEING ABLE TO
DO REASONABLY GOOD URINE PREPS.
THE FIRST THING WE DO IS ACETONE
PRECIPITATION.
SO WE TRY TO CRASH OUT THE
PROTEIN EARLY ON, WASH IT AS
MUCH AS WE CAN.
WE END UP HAVING TO DO SEVERAL
ROUNDS OF CLEANUP ON IT THOUGH
BEFORE WE ACTUALLY PUT A PROTEIN
INTO THE MASS SPECTROMETER.
WE'LL DO C-18 CLEANUP ON
PEPTIDES AND C-1 18 TRAPS AS
WELL.
WE'VE EXPERIMENTED AT DIFFERENT
STAGES OF PROTEIN LEVEL.
WE'LL DO A SERIES OF WASHES TOO.
WE'LL USE S-TRAPS FOR DIGESTION.
WE'VE BEEN EXPERIMENTS WITH SP-3
AS ANOTHER SOLUTION BUT FOR NOW
IT'S WORKING FOR US FOR THIS
PARTICULAR SET OF DATA.
>> [OFF MICROPHONE].
>> SO, URINE IS CHALLENGING TO
NORMALIZE BECAUSE OF VARIATION
IN CONCENTRATION.
FIRST THING WE DID, I MADE A SET
OF SAMPLES, LAB STANDARDS, THAT
ARE MY URINE THAT ARE -- I WAS
ACTIVELY TRYING TO LIKE DRINK A
BUNCH OF WATER OR DEHYDRATE
MYSELF TO MAKE US DEAL WITH THE
WORST POSSIBLE PROBLEMS OF THIS,
TO TRY TO FIGURE THIS OUT AHEAD
OF TIME, RIGHT?
RATHER THAN WORKING WITH
COMMERCIAL STANDARDS, THIS WAS A
WAY TO ACTUALLY MAKE A STANDARD
FOR WHAT WE CARED ABOUT WHICH
WAS TRYING TO DEAL WITH THIS
PARTICULAR PROBLEM OF
VARIABILITY.
AND SO FROM THAT PERSPECTIVE,
THE IMMEDIATE CRASH IS REALLY
IMPORTANT, ASSESSING A PROTEIN
CONCENTRATION AT THAT STAGE IS
IMPORTANT.
WE FEEL LIKE WE HAVE TO DO
PEPTIDE-LEVEL CONCENTRATIONS AS
WELL.
TO DEAL WITH THE NORMALIZATION
OF HOW MUCH PROTEIN WE'RE
ACTUALLY PUTTING ON THE MASS
SPECTROMETER, THAT'S THE BIGGEST
WAY TO DEAL WITH NORMALIZATION
BETWEEN THE SAMPLES.
THAT GETS US UP TO THE SAME
PROTEIN LEVEL.
THAT'S ANALOGOUS TO DOING AN
ASSAY TO SAY WHAT IS THE OVERALL
PROTEIN AND NORMALIZING TO THAT,
IT'S ANALOGOUS TO THAT.
>> [OFF MICROPHONE].
>> GOOD QUESTION THOUGH.
>> [OFF MICROPHONE].
>> PERFECT.
>> WE'LL NOT HAVE TIME TO
ENTERTAIN QUESTIONS FROM THE
ROOM.
SO NEXT TIME COME OVER HERE.
LET'S THANK BRIAN FOR COMING OUT 1976 01:14:22,124 HERE.