Table 1 Head-to-Head comparison of the fundamental characteristics defining of the IFCC-endorsed Reference Measurement Procedure (RMP) and the Designated Comparison Method (DCM) for apo(a)

General & Pre-analytical

Intended use

Higher order RMP as an essential part of the apo(a) traceability chain and future RMS

Designated Comparison Method for the future apolipoprotein RMS

Measurands addressed and units of reporting

Serum apo(a) (nmol/L)

Serum apo(a) (nmol/L)

Sample matrix

Serum

Serum

Automation

No, Manual

Yes, Semi-automated

Transition selection

Three transitions per peptide

Three transitions per peptide

Liquids applied in sample preparation

Buffer: 100 mM Ammonium Bicarbonate, pH 8.1

Reduction mix: 1.15 mmol/L TCEP, 0.40% (v/v) DOC

Buffer: 100 mM tris(hydroxymethyl)aminomethane, pH op 8.1

Reduction mix: 0.5 mM TCEP, 0.523% (v/v) DOC

Pre-digestion

LysC, 1:700 w/w LysC-to-protein ratio

None

Pre-digestion time

1 h, 37 °C

N.A

Digestion

Trypsin, 1:47 w/w Trypsin-to-protein ratio

Trypsin, 1:35 w/w trypsin-to-protein ratio

Digestion time

3 h, 37 °C

3 h, 37 °C

Main technology of sample purification

Solid phase extraction (off-line) with Oasis HLB 3 mg/well, eluted using 0.2 mL 80% MeOH

Solid phase extraction (off-line) with Oasis HLB 3 mg/well, eluted using 0.1 mL 55% MeOH

Proteotypic peptides of apo(a)

Apo(a)

LFLEPTQADIALLK

GISSTTVTGR

TPENYPNAGLTR

LFLEPTQADIALLK

GISSTTVTGR

LC–MS acquisition conditions

General LC setup

Agilent 1290 infinity II ultra-high performance LC system

Agilent 1290 infinity II ultra-high performance LC system

Guard column

Zorbax SB-C18

Zorbax SB-C18

Guard column geometry

2.1 × 5 mm, 1,8 µm

2.1 × 5 mm, 1,8 µm

Analytical column

Zorbax SB-C18

Zorbax SB-C18

Main column geometry

2.1 × 50 mm, 1,8 µm

2.1 × 50 mm, 1,8 µm

Mobile phase constituents

MeOH (HPLC grade), FA, Ultrapure water

MeOH (HPLC grade), FA, Ultrapure water

Flow Rate

0.2 mL/min

0.2 mL/min

Sample injection Volume

10 μL

10 μL

Ionization Mode

Positive

Positive

total running window

20 min

19 min

Mass spectrometer

Agilent 6495A triple quadrupole mass spectrometer

Agilent 6495A & 6495C triple quadrupole mass spectrometer

Gradient, generic description

A: 5% MeOH and 0.05% FA in water; B: 95% MeOH and 0.05% FA in water. Starting condition: A 92% -

Linear decrease to 82% A over 7 min—Linear decrease to 40% A at 15 min—Washing step: Steep decrease to 5% A at 15.1 min—Isocratic hold at 5% A until 17 min—Reequilibration: 3 min using starting conditions

A: 5% MeOH and 0.05% FA in water; B: 95% MeOH and 0.05% FA in water. Starting condition: A 95% -

Linear decrease to 67% A over 8 min—Linear decrease to 43% A at 12 min—Washing step: Steep decrease to 5% A at 12.1 min—Isocratic hold at 5% A until 16 min—Reequilibration: 3 min using starting conditions

Main MS ionization mode

Electrospray, positive polarity

Electrospray, positive polarity

Fragmentation

Collision induced dissociation

Collision induced dissociation

Run acceptance & quantitation

Internal Standard

In house (¹³C, ¹⁵N)R or (¹³C,¹⁵N)K SIL peptides

In house (¹³C, ¹⁵N)R or (¹³C,¹⁵N)K SIL peptides

Calibration concentrations

MCA 2013.2062

apo(a): 93.4 nmol/L;

MCA 2019.1564/2019.1565/2019.1566/2019.1568/2019.15611

apo(a): 17.1 – 41.2 – 93.4 – 270.7 – 9.9 nmol/L;

Type of calibration and calibration samples matrix

External protein calibration based on a native human serum sample with internal standard

External protein calibration based on native human serum samples with internal standard

Traceability

Indirect traceability to secondary reference material for apo(a): SRM-2B

Indirect traceability to secondary reference material for apo(a): SRM-2B

Data Analysis Software

Mass Hunter Workstation Quantitative/Qualitative Analysis software/Skyline

Mass Hunter Workstation Quantitative/Qualitative Analysis software

Interpretation of data

All transitions (both quantifying and qualifying) were evaluated individually

All transitions (both quantifying and qualifying) were evaluated individually